Characterization and identification of sources of rust resistance in Triticum militinae derivatives

Triticum militinae (2n = 4X = 28, AtAtGG), belonging to the secondary gene pool of wheat, is known to carry resistance to many diseases. Though some disease resistance genes were reported from T. timopheevii, the closest wild relative of T. militinae, there are no reports from T. militinae. Twenty-one T. militinae Derivatives (TMD lines) developed at the Division of Genetics, IARI, New Delhi, were evaluated for leaf and stripe rusts at seedling and adult plant stages. Eight TMD lines (6–4, 6–5, 11–6, 12–4, 12–8, 12–12, 13–7 and 13–9) showed seedling resistance to both leaf and stripe rusts while six TMD lines (7–5, 7–6, 11–5, 13–1, 13–3 and 13–4) showed seedling resistance to leaf rust but adult plant resistance to stripe rust and three TMD lines (9–1, 9–2 and 15) showed seedling resistance to leaf rust but susceptibility to stripe rust. Three TMD lines (2–7, 2–8 and 6–1) with adult plant resistance to leaf and stripe rusts were found to carry the known gene Lr34/Yr18. Ten TMD lines (7–5, 7–6, 9–1, 9–2, 11–5, 11–6, 12–12, 12–4, 12–8, and 15) with seedling resistance to leaf rust, showing absence of known genes Lr18 and Lr50 with linked markers requires further confirmation by the test of allelism studies. As not a single stripe rust resistance gene has been reported from T. militinae or its close relative T. timpopheevii, all the 8 TMD lines (6–4, 6–5, 11–6,12–4, 12–8, 12–12, 13–7 and 13–9) identified of carrying seedling resistance to stripe rust and 3 TMD lines (13–1, 13–3 and 13–4) identified of carrying adult plant resistance to stripe rust are expected to carry unknown genes. Also, all the TMD lines were found to be cytologically stable and thus can be used in inheritance and mapping studies.

Wild and related species of wheat carry enormous genetic variability that can be used in breeding programmes to broaden the genetic base of cultivated wheat.About half of the genes have been transferred to wheat through alien introgression [14][15][16][17] .Many wild species belonging to secondary and tertiary gene pools carry abundant genes resistant to biotic and abiotic stresses.However, they are still untapped because of the difficulty in making wide crosses and getting fertile F 1 seeds 16,18 .One such wild species, Triticum militinae, belonging to the secondary gene pool of wheat, carries enormous genetic variability for disease resistance.T. militinae is considered a spontaneous mutant of T. timopheevii 19 .However, another view is that it originated from an introgressive hybridization between T. timopheevii and T. cathlicum Nevski (T.persicum Vav.) 20 .T. timopheevii has immunity to many diseases, such as leaf rust, stripe rust, stem rust, powdery mildew, loose smut, karnal bunt and dwarf smut 21 .However, till now, not a single disease resistance gene has been reported from T. militinae.As such, five-leaf rust resistance genes (Lr18 22 , Lr50 23 , LrTt1 24 , LrTt2 25 and LrSelG12 26 ), four stem rust resistance genes (Sr36, Sr40, SrTt3 and Sr37) 27 and three powdery mildew resistance genes (Pm6 28 , Pm27 29 and Pm37 30 ) were reported to be transferred from T. timopheevii to bread wheat.Breeders have always been interested in exploring this untapped wild species to identify useful resistance genes.At the Division of Genetics, IARI, New Delhi, the wheat group has developed 44 introgression lines (ILs), named TMD lines (T.militinae Derivatives).They were developed by crossing bread wheat lines, Chinese Spring and NI5439 with Triticum militinae acc.No. 117001 and backcrossing the F 1 s to Chinese Spring for three generations and further selfing of BC 3 F 1 for seven generations.Earlier, Natraj et al. 2017 characterized three leaf rust resistance TMD lines (TMD6-4, TMD7-5 and TMD11-5) using SSR markers to identify the T. militinae introgression points in these three derivatives.In the current study, we have used 21 TMD lines (based on stability in their performance over the years) for screening at seedling and adult plant stages for leaf and stripe rusts.It gave us information on whether a particular derivative carries seedling or adult plant resistance or both.Also, the markers of known genes were used to screen these 21 TMD lines and to obtain initial knowledge of lines with unidentified sources of rust resistance.

Cytological analysis
Cytological analysis of twenty-one TMD lines showed the presence of 42 chromosomes (21 bivalents) in all the derivatives during meiotic metaphase I.The presence of 21 bivalents indicates the cytological stability of these T. militinae derivatives and the absence of any numerical aberration in them.This study also indicates the suitability of these TMD lines for inheritance and mapping studies.Representative pictures showing 21 bivalents in some of the TMD lines are presented in Fig. 1.

Screening of TMD lines for Lr18 and Lr50
Screening of TMD lines with linked marker Xwmc75 32 of Lr18 suggested the probable presence of Lr18 in 9 TMD lines (Table 2; Fig. 5).Apart from these, all other TMD lines showed absence of Lr18 in them.Out of these nine lines, two lines (TMD2-7 and TMD2-8) are identified as having adult plant resistance to both leaf and stripe rusts, four lines (TMD6-4, TMD6-5, TMD13-7 and TMD13-9) with seedling resistance to both leaf and stripe rusts and three lines (TMD13-1, TMD13-3 and TMD13-4) with seedling resistance to leaf rust but adult plant resistance to stripe rusts.T. militinae also showed a 180 bp band as of positive check T. timopheevii (Fig. 5), indicating the presence of Lr18 in T. militinae.Screening with Xgwm382 23 linked marker of Lr50 suggested the probable presence of Lr50 in eight T. militinae derivatives (Table 2; Fig. 6).Out of these, three lines (TMD2-7, TMD2-8, TMD6-1) are identified of having adult plant resistance to both leaf and stripe rusts, three lines (TMD6-5, TMD13-7 and TMD13-9) with seedling resistance to both leaf and stripe rusts and two lines (TMD13-1 and TMD13-3) with seedling resistance to leaf rusts but adult plant resistance to stripe rusts.

Discussion
Wild and related species of wheat carry enormous genetic variability that can be utilized in breeding programmes to broaden the genetic base of cultivated wheat.Triticum militinae, belonging to the secondary gene pool of wheat, carries enormous genetic variability for disease resistance.To date, no gene that confers rust resistance has been discovered in T. militinae.So, characterizing the resistance of T. militinae derivatives and knowing their cytological stability is important before using them in inheritance and mapping studies.T. militinae (A t A t GG;   www.nature.com/scientificreports/2n = 4X = 28) is a tetraploid with 14 pairs of chromosomes, while T. aestivum (AABBDD; 2n = 6X = 42) is a hexaploid with 21 pairs of chromosomes.The transfer of genes from T. militinae to T. aestivum is possible through homologous and homoeologous pairing.'G' genome of T. militinae has affinity to 'B' genome of wheat, which enable it to pair and exchange genomic regions 33 .Crossing of hexaploids with tetraploids produces sterility due to the production of univalents.With repeated backcrossing of F 1 to hexaploid parent, there is a reduction in the number of univalents and an increase in the number of bivalents, which in turn increases the fertility of backcross derivatives.An increase in fertility levels from BC 1 to BC 2 generations is observed in a cross between T. aestivum and Ae.cylindrica 34 .T. militinae derivatives showed 21 bivalents as they have been developed by three generations of backcrossing and seven generations of selfing.The TMD lines with unidentified leaf and stripe rust resistance genes can now be used for inheritance and mapping studies as they are cytologically stable.Evaluation of T. militinae derivatives for leaf and stripe rusts at seedling and adult plant stages and screening with gene based and linked markers of known genes provided insight into the distribution of known leaf rust resistance genes and their resistance pattern in T. militinae derivatives.Also, TMD lines with unknown rustresistance genes were identified for further studies.
Rest seven TMD lines with seedling resistance to leaf rust were found to be marker positive for Lr18, and some even showed the presence of Lr50 in them.Leaf rust resistance gene Lr18 provides seedling resistance to leaf rust but is temperature sensitive.It only provides effective resistance to its avirulent races at lower temperatures (18 °C) and becomes ineffective as the temperature goes beyond 25 °C36 .So, to confirm the presence of Lr18, these lines need to be tested at different temperatures, or it can be confirmed through test of allelism studies.Even the lines that showed the absence of Lr18 can be tested similarly to confirm its absence.Although identified in some lines, the leaf rust resistance gene Lr50 is not effective against leaf rust race 77-5 used in the current study.Also, virulence towards Lr50 existed in races of P. triticina even before its deployment in wheat cultivars 23 .So, Lr50 can provide durable resistance only when it is pyramided along with other effective resistance genes 23 .So, the resistance pattern of TMD lines in the current study is mostly based on seedling resistance gene Lr18, adult plant resistance gene Lr34/Yr18 and/or some unknown genes in T. militinae.
Adult plant resistance to leaf and stripe rusts in three TMD lines (TMD2-7, TMD2-8 and TMD6-1) and identification of APR gene Lr34/Yr18 with marker csLV34 indicates resistance to leaf and stripe rusts are because of APR genes Lr34 and Yr18 respectively.Of the three, two lines, TMD2-7 and TMD2-8, showed the presence of Lr18 but did not show any seedling resistance to leaf rust.Apart from these two, all other TMD lines (6-4, 6-5, 13-1, 13-3, 13-4, 13-7 and 13-9) identified carrying Lr18 showed seedling resistance.The susceptibility of lines TMD2-7 and TMD2-8, even with the presence of Lr18, may be due to the occurrence of recombination events between the gene Lr18 and its marker XWmc75, present at a distance of 1.2 cM 32 .So, before using any of these 21 TMD lines in further studies, an allelism test with genetic stocks carrying Lr18 is required.
Screening for stripe rust resistance identified 8 TMD lines with seedling resistance and 9 TMD lines with APR to stripe rust.Out of 9 TMD lines with APR for stripe rust, 6 TMD lines (2-7, 2-8, 6-1, 7-5, 7-6 and 11-5) were found to carry the known gene Yr18/Lr34 from the wheat cultivar Chinese Spring, used in their development.Three TMD lines (13-1, 13-3 and 13-4) with negative responses to the Yr18/Lr34 marker indicate the presence of unknown adult plant resistance genes for stripe rust from T. militinae.Out of 8 TMD lines with seedling resistance to stripe rust, 4 TMD lines (6-4, 6-5, 11-6 and 13-9) also showed the presence of known APR gene, Yr18/Lr34 while 4 TMD lines (12-4, 12-8, 2-12 and 13-7) showed its absence.Therefore, all 8 TMD lines may carry unknown/unreported stripe rust resistance gene(s) from T. militinae, as till now, not a single stripe rust resistance gene has been reported from both T. militinae and T. timopheevii.So, the lines with unknown seedling and adult plant resistance genes for stripe rust can be explored soon for mapping and transfer in superior genetic backgrounds.The lines with unknown seedling resistance genes for stripe rust but with known APR gene, Yr18/ Lr34, can be phenotyped in the seedling stage to tap the seedling stripe rust resistance gene while the other four (without Yr18/Lr34) can be tested at both the stages.
Except TMD 17, all other lines were found to carry resistance to either leaf or stripe rusts.This study also highlighted the presence of seedling and adult plant resistance genes in T. militinae derivatives, which can be used in future studies for mapping purposes and their utilization in breeding programmes to broaden the genetic base of wheat varieties.

Conclusion
The present study evaluated 21 T. militinae derivatives for leaf and stripe rusts at seedling and adult plant stages.This gave us information about lines carrying seedlings and adult plants' resistance to leaf rust, stripe rust, and leaf and stripe rust.To know whether the resistance in TMD lines is because of known genes or unknown genes, markers of two genes (Lr18 and Lr50) from T. timopheevii and one gene (Lr34/Yr18) from Chinese Spring were amplified.While ten TMD lines showed the probable presence of unknown genes for leaf rust resistance at the seedling stage, no lines with unknown genes for adult plant resistance to leaf rust could be identified.For stripe rust, while eight lines were identified as carrying unknown genes at the seedling stage, three were identified as www.nature.com/scientificreports/carrying unknown genes at the adult plant stage.These lines were also found to be cytologically stable so that they can be utilized in inheritance and mapping studies in future.

Materials and methods
The plant materials used in the current study are twenty-one derivatives of T. militinae, parental lines, T. militinae (acc.No. 117001), Chinese Spring and NI5439 used in developing these derivatives and the susceptible check Agra Local.The T. militinae acc.no.117001 was received in 1993 from the Institute of Plant Science Research, Norwich, Norfolk, England, Great Britain.Chinese Spring was used during the screening of TMD lines for leaf and stripe rust resistance and as a positive check for the gene Lr34/Yr18 during marker screening.The wheat genotypes NI5439, and Agra Local are two susceptible cultivars of India, susceptible to all three types of wheat rusts.While NI5439 and Agra Local were used as susceptible checks during screening for leaf and stripe rusts, only Agra Local was used when screening TMD lines for known markers.All the lines were screened with leaf rust pathotype 77-5 and stripe rust pathotype 110S119.In India, the leaf rust race 77-5 has remained most predominant for more than 20 years, but recently, the leaf rust race 77-9 became wider spread than 77-5 37 .In the case of stripe rust, races 110S119, 238S119 and 46S119 are the three most prevalent and widespread races with a maximum frequency of 110S119 during 2021-22 37 .The race 77-5 is virulent on leaf rust resistance genes Lr1, Lr2a, Lr2c, Lr3a, Lr10, Lr13, Lr14a, Lr15, Lr17, Lr20, Lr23 and Lr26 and is avirulent on Lr9, Lr18, L19, Lr24 and Lr28.The stripe rust race 110S119 is virulent on stripe rust resistance genes Yr2, Yr2+, Yr3, Yr4, Yr6, Yr7, Yr8, Yr9, Yr6(un) Yr7(un) and avirulent on Yr1, Yr5, Yr9 + and Yr10.To identify new sources of leaf rust resistance, markers of designated leaf rust resistance genes, Lr18 and Lr50 from T. timopheevii, were screened in TMD lines, where T. timopheevii was used as a positive control.TMD lines were also screened with Lr34/Yr18 markers, as the Chinese Spring was used as a parental line during their development.TMD lines were screened for leaf and stripe rusts at seedling and adult plant stages.As most of the TMD lines showed resistance to either leaf, stripe or both diseases, their cytological stability was also studied at the meiotic metaphase stage so that they can be used in inheritance and mapping studies.The list of TMD lines used in the current study and their pedigree is given in Table 1.All methods were performed following the relevant guidelines/regulations/legislation.

Analysis of cytological stability of T. militinae derivatives
To study the cytological stability of T. militinae derivatives, immature spikes of appropriate size were collected during the booting stage.Anthers containing pollen mother cells (PMCs) at meiotic metaphase I were identified, fixed in Carnoy's I fixative, and stored at 4 °C.For slide preparation, the anthers were hydrolyzed in a 1N HCl solution at 60 °C for 12 min.Following this, they were stained with Feulgen solution in the dark.Subsequently, slides were prepared using the squash method, utilizing 2% acetocarmine solution.

Screening for leaf and stripe rust resistance at the seedling stage
Screening for leaf and stripe rust resistance was carried out with pathotypes 77-5 and 110S119, respectively, in the glass house of the Division of Genetics.The screening experiments were repeated during 2018-19 and 2019-20.The screening materials included 21 TMD lines, their parental lines, T. militinae, Chinese Spring and NI5439 and the susceptible check Agra Local (AL).All the lines were sown in aluminium trays of 4 × 10 × 3 inches in size in the glass house at the Division of Genetics, IARI, New Delhi.Seedlings of ten days old with completely opened primary leaves were inoculated separately with leaf and stripe rust races.An inoculation mixture of concentration ̴ 6 × 10 5 spores/ml was prepared by adding urediospores in tap water.A drop of tween20 was added to the inoculation mixture to attain affinity and uniform spread of the urediospores on the leaf surface.After inoculation, the trays were kept in the incubation chamber under diffused light for 48 h and then shifted to glass house benches.Infection types on each introgression line were recorded 12-14 days after inoculation, following the standard scoring method 38 .

Screening for leaf and stripe rusts at the adult plant stage
For screening of TMD lines at the adult plant stage, all 21 TMD lines, along with their parental lines, were sown in leaf and stripe rust nurseries.In the rust nursery, each line was planted in 1 m rows, with infector rows planted after every 20 lines.Two rows of infectors were also planted in borders to have sufficient and uniform disease spread.Spores of leaf rust race 77-5 and stripe rust race 110S119 were sprayed in the respective rust nurseries as a suspension in water fortified with Tween20 (0.75 µl/ml) at an average concentration of 20 urediospores/ microscopic field (10x × 10x).Inoculum suspension was also injected into the last internode of the plant with the help of a 2 ml hypodermic syringe at the boot leaf stage in the field before the emergence of the boot 39 .Plant response was recorded into five infection types 40 .

Screening with markers of known genes in T. militinae derivatives
To know whether these TMD lines carry known or unknown rust resistance genes, markers of leaf rust resistance genes Lr18 and Lr50 were used to screen TMD lines.Here, T. timopheevii was used as a positive check for Lr18 and Lr50 and Agra Local and NI5439 were used as negative checks.Though, no stripe rust resistance gene was reported from T. timopheevii or T. militinae, a marker of Yr18/Lr34 was also used in screening, as Chinese Spring was used as one of the parents in the development of TMD lines, which is known to carry linked stripe and leaf rust resistance gene Yr18/Lr34.Details of molecular markers used in the screening are presented in Table 3.

Figure 4 .Table 2 .Figure 5 .
Figure 4. Screening of TMD lines and parental lines for the leaf rust resistance gene Lr34/Yr18 with the linked marker csLV34.Here, lines with arrow marks are positive for the leaf and stripe rust resistance gene Lr34/Yr18.

Figure 6 .
Figure 6.Screening of TMD lines along with parental lines for the leaf rust resistance gene Lr50 with the linked marker Xgwm382.Here, lines with arrow marks are positive for the gene. https://doi.org/10.1038/s41598-024-59902-x

Table 1 .
Pedigree and disease reaction of T. militinae derivatives for leaf and stripe rusts. S

Table 3 .
Details of markers used in screening of T. militinae derivatives.