Conversion of superior bread wheat genotype HD3209 carrying Lr19/Sr25 into CMS line for development of rust-resistant wheat hybrids

Hybrid development is one of the most promising strategies for boosting crop yields. Parental lines used to create hybrids must have good per se performance and disease resistance for developing superior hybrids. Indian wheat line HD3209 was developed by introducing the rust resistance genes Lr19/Sr25 into the background of popular wheat variety HD2932. The wheat line HD3209 carrying Lr19/Sr25 has been successfully and rapidly converted to the CMS line A-HD3209, with 96.01% background genome recovery, based on selection for agro-morphological traits, rust resistance, pollen sterility, and foreground and background analyses utilizing SSR markers. The converted CMS line A-HD3209 was completely sterile and nearly identical to the recurrent parent HD3209. Based on high per se performance and rust resistance, the study concludes that the derived CMS line A-HD3209 is promising and can be employed successfully in hybrid development.


Phenotyping of leaf rust resistance
The backcross progenies till BC 3 F 1 were screened under field conditions against the mixed pathotypes and showed resistance with infection type (IT) ranging from 0 to 5R.The leaf rust-resistant parent HD3209 was highly resistant.It displayed the fleck reaction (;) at 5% leaf area (5R), and susceptible parent A-365 produced a susceptible phenotypic response with ITs 40S against mixture of leaf rust pathotypes (Table 2).All the backcross progenies were resistant in all the generations as the recurrent parent was resistant, and the backcross progenies will be either homozygous or heterozygous resistant.The selected homozygous BC 4 F 1 plants were resistant, with no visible leaf rust infection in the field.The developed A-HD3209 with ITs 5R showed no visible leaf rust infection, indicating the effectiveness of the dominant alien Lr19/Sr25 genes from the recurrent parent.25  25  Furthermore, the recurrent parent HD3209 line showed seedling resistance for leaf rust against all four pathotypes of P. triticina used in the study with IT "0;" while the A-365 line was susceptible with IT "33+".The A-HD3209 has also displayed a similar disease reaction with an IT of 0; under glasshouse conditions at the seedling stage against all four pathotypes tested separately (Fig. 3).

Foreground and background analysis
All the backcross progenies of resistant recurrent parent with the dominant gene are supposed to be resistant as they may be homozygous or heterozygous resistant.Hence, it was essential to identify plants with homozygous resistant gene; otherwise, they may segregate for resistance in hybrids after crossing with restorer lines lacking resistance genes.Among the five BC 3 F 1 plants tested for foreground markers Xwmc221 and Xgdm67, four plants   www.nature.com/scientificreports/showed homozygous condition (Fig. 4).Among the plants homozygous for rust resistance, the best plant was selected based on agro-morphological traits and the BC 4 F 1 progenies from the same selected plant was further used.To identify polymorphic SSRs for background analysis, 947 SSR markers across the 21 chromosomes of bread wheat A, B, and D-genomes were screened for parental polymorphism between donor A-365 and recurrent parent HD3209.One hundred and thirty-eight markers displayed presence/absence and/or length polymorphism between A-365 and HD3209.The polymorphic markers representing each chromosome arm were further employed for background analysis of BC 4 F 1 plants (Fig. 5, Supplementary Fig. S1, Supplementary Table S1).These plants were also cross-verified for their phenotypic resemblance with their recurrent parent, HD3209 to minimize the plant-difference of CMS line A-HD3209.Background analysis was carried out using molecular markers in plants carrying target Lr19/Sr25 genes and sterile cytoplasm in BC 4 F 1 generations for each polymorphic locus of recurrent parent, HD3209-specific allele.In the BC 4 F 1 generation of A-365 × HD3209 plants, 96.01% of the genome of the recurrent parent was recovered based on background analysis using molecular markers (Fig. 6).Plants exhibiting higher background genome recovery (BGR) for recurrent parent were identified and analysed for agro-morphological similarity.

Agro-morphological similarity of A-line with recurrent parent
The A-HD3209 plants were tested for agro-morphological traits based on DUS testing guidelines and found to have almost complete similarity with recurrent parent HD3209, which will be further used as maintainer line, B-HD3209.The similarity with morphological traits indicated that the A-lines has recovered most genomic region from recurrent parent and have cytoplasm from donor parent with complete pollen sterility (Supplementary Table S2).

Discussion
In hybrid breeding, while it is pre-requisite to select heterotic combinations, it is also important to integrate disease resistance in parental lines to reduce yield losses due to different diseases.In this context, the present study aimed at transferring T. timopheevii-derived sterile cytoplasm from CMS line A-365 to a superior genetic background, HD3209, which also carries resistance to leaf and stem rusts.The wheat genetic stock HD3209 is a near isogenic line (NIL) of wheat variety HD2932, carrying leaf and stem rust resistance genes Lr19/Sr25 9 .Wheat variety HD2932 is a high yielding variety of the central and peninsular zones of India.The NIL of HD2932, HD3209 also performed very well in All India Coordinated Research Project (AICRP) trials 26 , based on which it was taken for conversion into a CMS/A-line.According to Singh et al. 27 , the 7D.7Ag translocation improved grain yield potential across the genotypes by 10-15%.Hence, it was widely used in conventional and markerassisted selection to incorporate leaf rust resistance.
The F 1 plant between A-365 and HD3209 was completely sterile and has been backcrossed with the recurrent parent.The spike of backcrossed F 1 plants have complete seed setting, which indicates typical stigma receptivity and standard seed setting in sterile plants on receiving sufficient pollen.Complete sterility and high seed setting on the crossing with pollen parent confirm the role of timopheevii cytoplasm in providing stable sterility and no undesirable effect on stigma receptivity.Timopheevii cytoplasm has been considered in many hybrid breeding programs and was advantageous over other cytoplasmic sources 20 .The backcrossed seeds were planted in field condition with the recommended package and practice.The seedlings were inoculated with mixed leaf rust pathotypes; as expected, all the seedlings were resistant.This happened because all the BC 1 F 1 seeds were either RR or Rr, as the recurrent parent was RR.
All the progenies were resistant in the backcross generations because of a homozygous recurrent parent carrying the dominant gene Lr19 for leaf rust resistance.The homozygous plants in BC 3 F 1 generation were identified with the help of co-dominant linked markers Xwmc221 (190 bp) and Xgdm67 (129 bp).These markers were helpful in backcrosses breeding programs, and the gene has been successfully incorporated in many varieties to improve disease resistance 9,28 .The selection of homozygous plants was essential; otherwise, heterozygous plants may segregate in susceptible progeny on crossing with restorer lines not carrying any resistance gene and hence can show a mixture of susceptible plants on commercial cultivation.The selection in backcross generations were made based on simple visual agro-morphological traits like plant height, leaf orientation, overall plant architecture and days to heading compared to recurrent parent HD3209.The five most similar plants were selected in BC 1 F 1 , BC 2 F 1 and BC 3 F 1 .The five selected plants of BC 3 F 1 were tested for the presence of a resistance gene in homozygous conditions, and among the plants homozygous, the best plant was selected.The BC 4 F 1 progenies and parents were further tested for rust resistance under controlled glasshouse conditions.The sterile plants were completely resistant, similar to the RP/B-line.The background analysis has shown the recovery of 96.01% of the  recurrent parent genome.Theoretically the average background recovery in BC 4 F 1 generation is 96.87%, but it is not always possible to capture that particular plant without any selection tools 29 .Phenotypic selection combined with marker-assisted backcrossing has been used successfully to develop NILs of wheat varieties HD2967 and HD2932 7,8 .The present results indicate that rigorous phenotypic selection in backcross generations can help in identifying plants with high recovery of the recurrent parent genome.Furthermore, the agro-morphological traits of the converted A-line were similar to recurrent parent.Additionally, the A-line has complete sterility and high seed setting on pollination, with the B-line indicating its likely usefulness in hybrid development depending on its heterotic combination with diverse restorer lines.

Conclusion
Since the green revolution, several attempts have been made to develop hybrids in wheat crops, mainly using a cytoplasmic male sterility (CMS) system.However, hybrids have yet to be developed, having higher yields with disease resistance.The present study has converted superior wheat genotype HD3209 carrying Lr19/Sr25 into CMS line A-HD3209 by crossing donor CMS line A-365 and recurrent parent HD3209.The converted CMS line A-HD3209 carrying Lr19/Sr25 has complete resistance to leaf rust disease and has agro-morphological traits similar to recurrent parent HD3209.Hence, CMS line A-HD3209 can be utilized as a potential seed parent and, thus, be the first step towards hybrid development with rust resistance in wheat.

Plant materials
HD3209, a rust-resistant line carrying Lr19/Sr25, has been used as the recurrent parent.The recurrent parent has been developed through the rapid transfer of rust resistance genes in a wheat variety HD2932 9 , which was released and recommended for the late sown condition of the central and peninsular zones of India.CMS line A-365 has been used as the donor parent for sterile cytoplasm.

Crossing and plan of conversion
The CMS line A-365 was used as a female parent and crossed with HD3209 as a pollen parent.The five F 1 plants were backcrossed with recurrent parent, HD3209.The 100 BC 1 F 1 seeds were grown, and five phenotypically selected plants were backcrossed with recurrent parent.The selection in BC 1 F 1 was performed based on resistance to leaf rust, the similarity of the plant with recurrent parent and complete pollen sterility.The five plants having the most significant phenotypic similarity with recurrent parent, complete pollen sterility and rust resistance were selected and further backcrossed.Twenty seeds from each of the five BC 1 F 1 plants were bulked, and 100 BC 2 F 1 plants were grown.Similar criteria were used in BC 2 F 1 , and the five best plants were selected.Again, 100 BC 3 F 1 plants were grown from the bulk of five selected plants from the previous generation.The five selected BC 3 F 1 plants having complete sterility were identified for resistance genes in homozygous conditions through foreground analysis, and further the phenotypically best plant was selected for utilization in generation advancement and development of A-line (Table 1, Fig. 1).

Evaluation for rust resistance in segregating backcross generations
The parental lines and backcrossed progenies under different generations till BC 3 F 1 were tested with a mixture of Puccinia triticina pathotypes (77-5, 77-9, 12-5 and 104-2) under field conditions.Pure inoculum (uredospores) of pathotypes obtained from the ICAR-Indian Institute of Wheat & Barley Research, Regional Station, Flowerdale, Shimla were initially multiplied on susceptible Agra Local cultivars in a glass house at the Division of Genetics, ICAR-IARI, New Delhi.Under the field condition, backcrossed plants and the infector were evaluated for leaf rust at the adult plant stage.A mixture of susceptible cultivars, such as Agra Local, Kharchia Local and A9-30-1, were used as infector to spread the disease uniformly under field conditions.Approximately 55 days old plants were sprayed with a suspension of mixed inoculums prepared in water with a droplet of Tween-20.After 10-15 days of inoculation, individual plants of backcrossed segregating plants grown with infector rows were scored following a modified Cobb scale 30 .

Molecular characterization for foreground and background analysis
To confirm the presence of the Lr19/Sr25 genes in homozygous condition (BC 3 F 1 ) and background recovery (BC 4 F 1 ), the DNA was extracted from the selected plant using the CTAB method and the quality and quantity of DNA was confirmed on 0.8% agarose gel and Nanodrop spectrophotometer.The PCR reaction for SSRs was performed in a reaction volume of 10 μl, which included 4 μl of 2 × GoTaq PCR Master Mix (Promega, #M7122), 1 μl of each primer (5 pmol/μl), 2 μl of nuclease-free water, and 2 μl of 25 ng/μl gDNA (50 ng) in 96-well PCR plates with a thermal seal in an Eppendorf thermal cycler.A thermal profile of 4 min at 94 °C (initial denaturation), followed by 35 cycles of the 30 s at 94 °C (denaturation), 30 s at 50-60 °C (varies according to primer annealing temperature), 30 s at 72 °C (primer extension), and final extension at 72 °C for 10 min was used in a PCR machine for SSR marker amplification.On a 3.5% agarose gel, the amplified products were resolved and observed using a UV transilluminator Gel Documentation System (G: Box, Syngene).Recurrent parent genome (RPG) recovery was determined as the number of homozygous loci matching the recurrent parent plus half the number of heterozygous loci, divided by the total number of polymorphic SSR markers loci and multiplying the resultant by 100 23 .
Polymorphic SSR markers, i.e., Xgdm67 and Xwmc221, closely linked to the Lr19/Sr25 genes on chromosome 7DL of wheat, were used for foreground analysis in BC 3 F 1 to confirm the presence of the genes in homozygous condition 9,28 .The background analysis was carried out on the bulked sample of 10 plants of BC 4 F 1 from the selected best BC 3 F 1 plant found to be homozygous for the resistance gene.The parental polymorphism between

Figure 1 .
Figure 1.Systematic backcross breeding scheme for development of CMS line A-HD3209.

Figure 6 .
Figure 6.Graphical genotypes (GGT) describing recurrent parent genome recovery across the 21 chromosomes of derived CMS line A-HD3209.

Table 1 .
The details of backcrossed plants grown and selected for generation advancement.a Number indicates the plants bulked to grow 100 plants at given generations.

Table 2 .
Infection type (ITs) of leaf rust incidence level recorded in the parental lines and selected homozygous BC 4 F 1 plants. S.