Location and Identification on Chromosome 3B of Bread Wheat of Genes Affecting Chiasma Number

Abstract

Understanding meiotic crossover (CO) variation in crops like bread wheat (Triticum aestivum L.) is necessary as COs are essential to create new, original and powerful combinations of genes for traits of agronomical interest. We cytogenetically characterized a set of wheat aneuploid lines missing part or all of chromosome 3B to identify the most influential regions for chiasma formation located on this chromosome. We showed that deletion of the short arm did not change the total number of chiasmata genome-wide, whereas this latter was reduced by ~35% while deleting the long arm. Contrary to what was hypothesized in a previous study, deletion of the long arm does not disturb the initiation of the synaptonemal complex (SC) in early meiotic stages. However, progression of the SC is abnormal, and we never observed its completion when the long arm is deleted. By studying six different deletion lines (missing different parts of the long arm), we revealed that at least two genes located in both the proximal (C-3BL2-0.22) and distal (3BL7-0.63-1.00) deletion bins are involved in the control of chiasmata, each deletion reducing the number of chiasmata by ~15%. We combined sequence analyses of deletion bins with RNA-Seq data derived from meiotic tissues and identified a set of genes for which at least the homoeologous copy on chromosome 3B is expressed and which are involved in DNA processing. Among these genes, eight (CAP-E1/E2, DUO1, MLH1, MPK4, MUS81, RTEL1, SYN4, ZIP4) are known to be involved in the recombination pathway.

Keywords: 3D-SIM; chiasmata; cytogenetics; deletion bin; meiosis; recombination; synapsis; wheat.

Figure 1

Meiotic behaviour of pollen mother cells (PMCs) of wild-type, Nulli 3B and ditelosomic Dt3BS Chinese Spring lines. Wild-type cells (a) exclusively show ring bivalents (ri) at metaphase I. Nullisomic 3B cells (b) exhibit a range of ring or rod (ro) bivalents and rare univalents (u), while ditelosomic Dt3BS cells (c) contain ring-bivalents and many rod bivalents (ro) and univalents (u), suggesting loss of obligate crossovers. Scale bar = 10 µm.

Figure 2

Average numbers of chiasmata (red crosses) for the various Chinese Spring lines: wild-type (CS), ditelosomic (Dt3BS and Dt3BL), deletion lines (3BS-xx or 3BL-xx for short and long arms respectively), hybrids between CS and Dt (F1xxx) and nulli-tetrasomics. ***: p < 0.001.

Figure 3

Comparison of synapsis in CS, 3BL7 and Dt3BS by 3D super resolution imaging. Chromosomes axes are labelled with ASY1 (magenta), and ZYP1 (green) is used to follow synapsis. In CS (a–d), synapsis starts at one side of the nucleus (white star; a) and progressively brings the homologues together at zygotene (b,c). Chromosomes are fully synapsed at the pachytene/diplotene stage (d). In 3BL7 (e–h), synapsis resembled that of the wild type of CS. In Dt3BS, the initiation of synapsis (i) and the progression at zygotene (j) are similar to CS. However, during zygotene, ZYP1 forms polycomplexes (white arrow; k) during synapsis, suggesting a difficulty to polymerize the central element of the synaptonemal complex, although chromosomes seem aligned with a dotty synapsis at pachytene (l). Scale bar = 5 µm.

Figure 4

Homologous chromosome pairing and segregation at Metaphase I and Anaphase I in lines CS (a,d), 3BL7 (b,e) and Dt3BS (c,f). Homologous pairing was monitored using the 45S-rDNA probe. All lines exhibit correct homologous chromosome pairing as shown by the localisation of two 45s signal (red, yellow arrows) per bivalent (DAPI blue). Homologous chromosomes halve at anaphase I in both CS (d) and 3BL7 (e), with occasional chromosome bridges (white arrows) in 3BL7. We could not find a similar stage for Dt3BS. Moreover, Dt3BS chromosomes were “fuzzier” and stickier at Metaphase I (f) than in the other lines, making chromosome spread for 3D DNA in situ hybridization in this line more challenging. Scale bar 10 µm.

Figure 5

Level of expression (FPKM values; average of the four stages and two replicates) in meiotic tissues [55] of the different homoeologous copies (A: orange; B: yellow; D: green) of genes known as being involved in recombination in model species and located on chromosome 3B.