GISH and FISH analysis
GISH was used to analyse the number of chromosomes and to determine the karyotype of WAT650l using A. cristatum Z559 genomic DNA as GISH probe. The results indicated that WAT650l is a translocation line with 42 chromosomes. Among these, a pair of chromosomes had an obvious red signal at the terminus, and the other 40 chromosomes displayed completely blue 4,6-diamidino-2-phenylindole (DAPI) signals. These results confirmed that part of the A. cristatum chromatin was translocated into Fukuho(Fig. 1a).
To further verify the WAT650l karyotype, the oligonucleotide probes Oligo-PTa535-1 (red) and OligopSc119.2-1 (green) were used for FISH analysis. Compared with the Chinese Spring karyotype map, WAT650l had 14 complete chromosomes of the A subgenome and D subgenome, and 12 complete chromosomes of the B subgenome(1B, 2B, 3B, 4B, 6B, 7B) have been proven already. In addition, after combining the GISH results, we confirmed that the 5BS terminal segments of Fukuho translocated with the 6P chromosome of Z559 (Fig. 1b). In conclusion, WAT650l is a novel 5BS·6P small segment translocation line.
Chromosomal location of introgressed chromatin in wheat–A. cristatum translocation line WAT650l
A total of 36 molecular markers from 31 segments of chromosome 6P were used to identify the segments from A. cristatum in WAT650l (Table 1). A. cristatum Z559, 4844-12, translocation line PB260 (harbouring 6PL14-17 bin) and deletion line del10c (harbouring 6PL1-13 bin) were used as positive controls, while Fukuho and Gaocheng 8901 were used as negative controls.
The results showed that all 36 markers could amplify specific bands in 4844-12. These bands were absent in the Fukuho and Gaocheng8901, and PB260 and del10c harbour bins 6PL (14-17) and 6PL (1-13), respectively, were confirmed in the same way as previously reported. All markers of the 6PL13 bin and two markers (Agc10741 and Agc68493) of the 6PL12 bin amplified specific bands in WAT650l, but two markers, namely, Agc9467 and Agc22320, from the 6PL12 bin and the other markers from the 6PS1-6PL11 bin failed to produce specific bands in WAT650l (Fig. 2a ~ 2i). Therefore, WAT650l harboured all the chromatin of the 6PL (13-17) bin and part of the chromatin of the 6PL12 bin. At the same time, three 5BS molecular markers, including two molecular markers designed using Chinese Spring reference genome sequences and the wheat-SSR marker Xgwm234 were used to check the integrity of the 5BS chromosome in WAT650l (Table 1). Compared with the genotyping results of Fukuho and 4844-12, the three markers revealed no specific bands in WAT650l through polypropylene gel electrophoresis (Fig. 2j, 2k, 2l). These results further confirmed that chromosome 6PL (~12-17) bin of A. cristatum translocated with the 5BS terminal chromosome of Fukuho in WAT650l.
Evaluation of WAT650l agronomic performance
The agronomic performance of WAT650l and its backcross parent Fukuho was investigated during the growing seasons of 2020-2021 and 2021-2022 in Xinxiang, Henan Province. The results showed that the average flag leaf length, plant height and spike length of WAT650l significantly increased by 4.59 cm, 16.73 cm, 1.14 cm, respectively, compared with those of Fukuho. In addition, through t test analysis, the GNS, KNS, SNS, and TGW of WAT650l increased by 14.46 and 11.94, by 1.26 and 0.66, by 2.41 and 4.67, and by 4.88 and 2.33, respectively, compared with those of Fukuho during the two years (P value < 0.01). However, there was a significant decrease in the number of effective tillers compared with the number for Fukuho (Table 2, Fig. 3a, 3b, 3f). The spike structure of WAT650l was also observed. We found that the WAT650l spikelets were more fertile, and most of the spikelets developed in balance on the whole, resulting in the spikes appearing ‘stick’-shaped rather than ‘spindle’-shaped like Fukuho (Fig. 3c, 3d, 3e).
To further verify the genetic benefits of 6PL terminal segments to the GNS and TGW of WAT650l, the yield traits (including effective tillers, weight per plant, KNS, GNS, SNS and TGW) of the plants composing the BC4F2 and BC5F2 populations were analysed. A total of 36 positive plants and 24 negative plants in the BC4F2 population and 108 positive plants and 93 negative plants in the BC5F2 populations were identified by the A. cristatum repeat sequence markers Acpr3a and Acpr7. Compared with negative plants, the positive plants in the BC4F2 population presented increases in SNS, KNS, GNS and TGW by 1.34, 0.33, 14.29 and 7.12, which were increases of 6.51%, 6.95%, 21.08% and 21.21%, respectively; the positive plants of the BC4F2 population presented a 3.15 decrease in the number of effective tillers, which was a decrease of 22.04% compared with that of the negative plants. Similarly, compared with negative plants, the positive plants in the BC5F2 population presented increases in SNS, KNS, GNS and TGW of 1.47, 0.55, 13.86 and 1.50, respectively, which were increases of 7.18%, 11.88%, 20.41% and 4.85%, respectively, and the positive plants of the BC5F2 generation presented a 2.32 decrease in the number of effective tillers, which was a decrease of 15.97% compared with that of the negative plants (P value < 0.01). However, there was no significant difference in weight per plant between the positive plants (17.47 g) and negative plants (19.21 g) in the BC5F2 genetic population (P value = 0.1022) (Table 2, Fig 4a, 4b). In addition, the segment from WAT650l was introgressed into KN199, the BC1F1 plants showed obvious high-yield traits, and the SNS, KNS, GNS and TGW increased by 2.18, 0.78, 11.75 and 11.25, respectively, which were increases of 10.71%, 18.48%, 19.19% and 30.44%, respectively (P value < 0.01) (Table 2). Therefore, the high-yield trait of WAT650l could stably inherited, and introgression of 6PL terminal segments from A. cristatum into Fukuho altered the spike development process and simultaneously increased the GNS and TGW.
Chromosome mapping of the loci responsible for increased GNS and high TGW within the 6PL terminal region
Previously, a large number of 6P translocation lines and deletion lines carrying different-sized chromosomes were generated by radiation-induced mutations, and a locus responsible for increased GNS was located on 6PL (0.27-0.51) via deletion line mapping. In addition, some translocation lines carrying 6PL terminal segments showed increased GNS; this was the case for the 3BS·6PL (14-17) translocation line PB260 and the 3AL·6PL (16-17) translocation line WAT642a, which were generated previously (Fig. 5). Genetic population analysis confirmed that, compared with the negative plants, the positive plants in the PB260 BC6F2 population presented 0.29 and 6.55 increases in KNS and GNS, respectively, which were increases of 6.07% and 9.83%, respectively. Similarly, compared with the negative plants, the positive plants in the WAT642a BC6F2 population presented increases in KNS and GNS equal to 0.36 and 13.20, respectively, which were increases of 7.47% and 19.68%, respectively (P value < 0.01). However, no significant differences in SNS or TGW were observed between the positive plants and negative plants of the PB260 and WAT642a BC6F2 populations (Table 2, Fig. 4c, 4d). Taken together, these results showed that three translocation lines, PB260, WAT642a and WAT650l, carried the common segment from 6PL (16-17) and presented increased KNS and GNS (Fig. 6); on the other hand, the segment from 6PL (14-17) did not contributed to increased SNS and TGW. Therefore, the loci responsible for increased KNS and GNS were mapped to 6PL (16-17), and the loci corresponding to high SNS and TGW were mapped to 6PL (12-13) (Fig. 7).