CMS Lines Research Articles

Assessment of Combining Ability for Yield and Associated Traits in Sunflower (Helianthus annuus L.)

The study was carried out at the Oilseeds Research Station, Latur. During kharif 2023, crosses were produced and during Rabi 2023–24, evaluation was conducted to assess the combining ability for seed yield and component traits in parents and hybrids in sunflower. In this study, 48 F1 hybrids were produced through Line x Tester mating design during Kharif 2023 using six CMS lines (DSF-2 A, CMS 148A, CMS 47A, CMS 597A, CMS 103A, CMS 112A) and eight testers (RHA-1055, EC-601951-1, EC-601924, R-274, LTRR-341, AKSF-15R, BK-R-1, R-856). These hybrids were assessed using a Randomized Block Design with two replications at the Oilseeds Research Station in Latur during Rabi 2023–24, together with their 14 parent lines and two commercial checks (LSFH-171 and KBSH-44). The observations were recorded on five randomly selected plants and the average value of these data was calculated for ten quantitative attributes. The analysis of combining ability (GCA and SCA) effect was came out by using the Line x Tester analysis method suggested by Kempthorne [1]. The significance of GCA and SCA effects was determined at the 0.05 and 0.01 level using the t-test [2]. All characteristics had substantial significant variance due to line x tester effects. The existence of non-additive gene action for days to maturity, head diameter, seed filling, 100 seed weight and seed yield per plant were recorded. The per cent contribution of testers towards total variance was greater than lines for all the characters except plant height. Among the parents, two lines CMS-597 A and CMS-47 A and four testers RHA-1055, EC-601924, R-274 and AKSF-15R were found to be superior general combiners for seed yield per plant and most of the yield contributing traits. On the basis of mean performance, SCA effects of crosses and GCA effects of the parents, the crosses viz., CMS-112 A x AKSF-15R, CMS-148 A x RHA-1055, CMS-597 A x R-856, CMS-47 A x R-274, CMS-597 A x LTRR-341, CMS-112 A x EC-601924 and CMS-597 A x EC-601951-1 recorded high SCA effect with low x high, low x high and high x high GCA status of parents for seed yield per plant.

Effects of row ratio and plant spacing for enhancing hybrid rice seed production in the tropics

The seed yield production determines the success of the development of hybrid rice. Here, an effective and efficient cultivation technology for producing hybrid rice seeds is optimized through spacing and row ratio management. This study aimed to evaluate the effects of spacing and row ratio of restorer (R) : CMS (A) on F1 hybrid rice seed production. The experiment was conducted at the Indonesian Center of Rice Research's experimental field in the dry season of 2016, using parental lines HIPA18 hybrid rice, i.e., CMS: IR79156A line and Restorer: IR53942 line. The experiment was laid out in a strip-plot design replicated three times. Two factors were studied, i.e., plant spacing between CMS lines and row ratio R by A-lines. The results indicated that the highest HIPA18 seed production was obtained from 20 cm x 20 cm spacing of CMS with row ratio 2R:14A, i.e., 3.62 tons ha-1. Plant spacing and row ratio interactions were improved tillers number per plant of restorer and F1 seed yields. Keywords: A-lines; CMS; F1 hybrid rice; R-lines

Comparison of Blast, Bacterial Leaf Blight and Brown Plant Hopper Introgressed Lines for Yield and Morphological Traits Over Original Line

Rice as a staple food for very large population suffers from various biotic and abiotic stresses. Among the biotic stresses, Blast, Bacterial leaf blight and Brown plant hopper (BPH) are considered to most potential threats that significantly affect the rice productivity. The use of chemical usage for controlling these diseases and pest attacks is not environmentally friendly and is expensive. Using Molecular Marker assisted backcross breeding program we have improved one of our hybrid parental line with 5 genes (xa13, Xa21, Pi54, Bph20 and Bph21). We have developed 15 near isogenic lines having similar agronomical characters as of recipient parent, they are promising for their direct induction in breeding program. Our results indicated that out of fifteen lines, Six lines i.e. GK 101-12, GK 101-15, GK 101-9, GK 101-5 and GK 101-2 out-performed in all the three locations tested, they had not only have very high level of resistance to BLB, Blast and BPH but also showed significantly higher yield compared to susceptible check as well as recipient parent. These lines may be used for improvement of our existing hybrid and also for developing of new hybrids with other CMS lines. These lines are also useful for developing new set of restorer lines through (R x R) and also useful for introgression of wide spectrum of resistance to the varieties/hybrid.

Enhancing yield and oil content in oilseed rape hybrids: Insights from line × tester and SIIG approaches.

The production of high-oil-yielding hybrid varieties is a primary objective in oilseed rape (Brassica napus L.) breeding programs. Biometric genetic experiments such as line × tester provide valuable insights into the genetic structure of traits associated with high oil yield. In this study, 21 winter hybrids of oilseed rape were evaluated, which were generated by crossing three restorers with seven CMS lines. The experiment was conducted using a line × tester experiment based on a completely randomized block design. Phenological, agronomic, yield, and oil yield components were assessed in this study. The ideal genotype selection index (SIIG) methodology was also employed to identify superior hybrids based on all studied traits simultaneously. Significant differences were observed between the obtained hybrids and the check cultivars. Heritability analysis revealed that phenological traits were primarily controlled by additive effects, while agronomic and qualitative traits were mainly influenced by non-additive gene effects. Both broad-sense and narrow-sense heritability exhibited a wide range, underscoring the importance of genetic variance. Notably, the hybrids T1 × L5, T1 × L6, and T3 × L1 showed significant specific combining ability values of 394.74, 541.73, and 1236.79, respectively, making them the top specific combinations for increasing seed yield. Based on the SIIG index, hybrids T3 × L1, T1 × L5, T1 × L3, and T2 × L3 emerged as high-oil-yielding hybrids with desirable agronomic traits. The identified superior hybrids by line × tester and SIIG approaches hold promise for the development of high-yielding oilseed rape cultivars with desirable agronomic traits in oilseed rape breeding programs.

Identification of SSR markers linked to new fertility restoration trait in sorghum (Sorghum bicolor (L.) Moench) for A4 (maldandi) male sterile cytoplasm

AbstractIdentification of markers associated with fertility restoration (Rf) genes is essential because they can streamline the breeding of new CMS lines and production of commercial hybrid seeds. Therefore, in the present study, F2 populations (M31‐2A × DSMR 8) was utilized to identify markers linked to Rf loci on maldandi (A4) cytoplasm through bulk segregant analysis (BSA). The F2 population was analysed for seed set percentage. Chi‐square (χ2) analysis showed that the fertility restoration trait followed expected digenic ratio. By BSA, simple sequence repeats (SSRs) markers, namely, Xtxp 34 and Xtxp 69 located on chromosome 3 and SB 3956 and Xtxp 312 located on chromosome 7, showed clear polymorphism between two groups of fertile and sterile bulks. The genomic region harbouring Rf locus on chromosome 3 (2.61 Mbp) predicted to encode five pentatricopeptide repeat (PPR) genes whereas, on chromosome 7, the gene SORBI_3007G047400 predicted to encode MYB (myeloblastosis) domain containing proteins. These predicted genes could be the candidate for restoring fertility on A4 cytoplasm. This finding will be fundamental in the production and rapid selection of novel restorer lines.

Combining ability for various parameters in sunflowers as a classical breeding approach for superior hybrid development

An experiment for estimating the combining ability in sunflower for various agronomic traits and oil content was carried out in 2021-22 at Punjab Agricultural University, Ludhiana, in which 91 hybrids were studied along with their parents, i.e., seven CMS lines and 13 high oleic testers. Significant positive and negative variations were noticed in GCA and SCA for characters like days to flowering initiation, fifty per cent flowering, plant height, head diameter, seed yield per plant and oil content. Dominant gene action was predominant for all these characters and high SCA was observed for high GCA × high GCA for flowering initiation, head diameter, and seed yield per plant and low GCA × high GCA for fifty per cent flowering, plant height and both high GCA × high GCA and low GCA × low GCA for oil content.

Maintainer/restorer identification for different CMS lines in sunflower (Helianthus annuus L.)

Thirty one uniform and stable germplasm accessions of economic importance of sunflower were crossed with five cytoplasmic male sterile lines viz., CMS-234A, CMS-17A, CMS-338A, CMS-851A and ARM-243A in a Line x Tester fashion to study their maintainer or restorer reaction during kharif-2012 in a Randomized Block Design in two replications. The accessions TSG-22, TSG-24, ID-7, SCG-37, RHA-464 and ID-25 acted asrestorersfor allfive CMS lines. It was found that ID-33 of the restorer for ARM-243A was maintainer for other CMS lines. The use of stable germplasm with restorer behaviour in heterosis breeding programmes and those with maintainer behaviour in the development of new CMS lines through conversion has been suggested. The identification of new restorers to the good combiner CMS sources assembled should receive priority for hybrid synthesis.

Correlation and Path Coefficient Studies for Grain Yield and Yield Components in Rice (Oryza sativa L.)

The current research was conducted with an objective to study the correlation between different traits and path coefficient analysis in rice. Four CMS lines were crossed with eight elite restorers in a Line × Tester design and the resultant 32 hybrids were evaluated along with the parents and two checks in a randomized block design with three replications during the rabi season of 2022. The data was collected on 13 quantitative characters. The results revealed that there is a significant variability in the material studied. The character association studies indicated that the trait, grain yield per plant had significant positive genotypic correlation with panicle length, number of productive tillers per plant, number of grains per panicle, 1000 grain weight, 50% flowering, plant height, kernel length, kernel breadth and head rice recovery. The path analysis studies revealed that panicle length was the major contributor for grain yield per plant followed by plant height, number of grains per panicle, days to 50% flowering, 1000-grain weight, kernel length, number of productive tillers per plant, head rice recovery and kernel breadth. These characters showed direct positive effects for grain yield per plant. The above characters can be used directly as the selection criteria in rice yield improvement programmes.

B × R cross de rived parental line development using breeding value: A new model for hybrid rice parental line development

Three-line hybrid rice system is the most successful and widely practiced method around the world. Hybrid rice breeders have used B × B, A × R and R × R (R = Restorer line, B = Maintainer line, A = CMS line) scheme of parental line improvement frequently and avoided B × R and R × B scheme. As a result, female parents lack the genetic diversity carried by R lines. But B × R and R × B mating have great potential to produce high value parental lines of hybrid rice and overcome the limitation of the previous approach. We have demonstrated a new method for three-line hybrid system to minimize the barrier of crossing in parent selection for developing new elite maintainers and restorers. Parental combinations were selected based on breeding value of the genotypes. Breeding values were estimated based on ancestor, pedigree information and yield data of 74 test genotype to select parents for restorer and maintainer line improvement. This new protocol allows (B × R), (R × B) (R × Elite) and (B × Elite) improvement technique to bring out high yielding diverse B and R lines. This B line will be used for developing new A line in the genetic background of B line. Doubled haploid and RGA i.e. rapid generation advance tools of breeding will save the precious time and reduce breeding cycle length; and large population size will increase selection accuracy. We have predicted the genetic gain in parental line development for four parental cross using the studied 74 genotypes for doubled haploid and rapid generation advance methods. Our objectives were to demonstrate the new breeding approach plus breeding value and positive dominant gene effect-based parent selection strategy. We are hopeful about the new method that hybrid rice breeders across the world will extract benefit utilizing the new methodology of hybrid rice parental line development.

Pollination Potentiality of Honey Bee (Apis cerana Fab.) in Sunflower Hybrid (DRSH-1) Seed Production

The study was conducted at Main Agricultural Research Station, UAS, Raichur during rabi 2020-21 to utilize honey bees in sunflower hybrid seed production. Totally, thirty species of floral visitors were recorded on parental lines of sunflower hybrid (DRSH-1), among them 15 were hymenopterans, 7 lepidopterans, 5 coleopterans and 3 dipterans. Apis cerana was predominant pollinator (17.91±6.72 bees/ 5 capitula/ 5 min) on CMS parental line (ARM-243), followed by A. dorsata and least was A. florea. The peak foraging activity of A. dorsata was recorded at 0900-1000 hr with 24 bees, A. cerana recorded two peak foraging activity, one at 1100-1200 hr (29 bees) and another at 1600-1700 hr (18 bees) and A. florea recorded at 1000-1100 hr (10 bees). The maximum Shannon-Wiener index of diversity (H=1.32) of floral visitors on CMS line under open condition was recorded at 0800-0900 hr of the day and least (H=0.84) was at 1600-1700 hr. In case of fertility restorer line (6D-1) maximum Shannon-Wiener index was recorded at 1600-1700 hr (1.24). A. cerana under caged condition (three colonies per acre) without pollen load on CMS line were recorded highest mean number of nectar foragers (65±22.60 bees), followed by A. dorsata, A. cerana and A. florea foragers from natural colonies in open pollination condition. Whereas, highest mean number of nectar foragers (42.25±23.59) with pollen load in A. cerana under caged condition (three colonies per acre) were recorded, followed by A. dorsata, A. cerana and A. florea. Nectar foragers with pollen load on CMS line play an important role in transfer of pollen grains from restorer line to CMS line and carry out effective pollination. A. florea with and without pollen load spent more foraging duration (13.17 and 25.20 sec/capitulum respectively) on CMS line, followed by A. cerana and least in case of A. dorsata in open pollination. Bees without pollen load spent significantly greater nectar foraging duration as compared with bees with pollen load, both in caged and open pollination situations. Significantly highest seed yield per hectare (839.53 kg), per cent filled seeds (95.80 %), test weight (5.41 g), volume weight (46.41 g/100 ml), kernel percentage (66.09 %), highest kernel to husk ratio (1.94) and highest seed quality and associated parameters were recorded in hand + open pollinated plots, followed by the A. cerana @ 3 colonies per acre treated plots, hand pollination, open pollination and least seed quality parameters were recorded in control treatment.

Studies on genotype x environment interactions of sunflower hybrids (Helianthus annuus L.)

Twenty eight hybrids derived from four CMS lines and seven restorer lines along with two checks were evaluated in randomized block design at three locations for G x E interactions and stability parameters. Stability analysis revealed that genotype x environment interactions were highly significant for all the eleven traits which indicated differential response of genotypes to varied environments. Cross combinations SVCMS-1 x SVSR-3, SVCMS-1 x SVSR-5, SVCMS-2 x SVSR-2, SVCMS-2 x SVSR-4, SVCMS-3 x SVSR-2, SVCMS-3 x SVSR-3, SVCMS-3 x SVSR-7, SVCMS-4 x SVSR-7 and check Phule Raviaj had significantly higher mean than population mean and regression coefficient less than unity which indicated their stable and wide adaptability to poorenvironment, while, hybrids, viz., SVCMS-1 x SVSR-7, SVCMS-2 x SVSR-3, SVCMS-2 x SVSR-6, SVCMS-3 x SVSR-1, SVCMS-3 x SVSR-4, SVCMS-3 x SVSR-5, SVCMS-3 x SVSR-6, SVCMS-4 x SVSR-1, SVCMS-4 x SVSR-5 SVCMS-4 x SVSR-6 and check LSFH-171 were with high mean, regression coefficient more than unity (bi>1) with non-significant least deviation from regression line indicating their specific adaptation to favorable or rich environments.

Molecular Marker and Test Cross Information Aid Selective Advancement of F4 Generation of CB174R/Azucena: An inter Sub-specific Cross in Rice for Restorer Development

The present investigation was aimed to develop restorer lines for three-line hybrid rice using indica/tropical japonica derivatives to exploit the inter sub-specific heterosis. From 75 F4 families of CB174R/Azucena, two plants were randomly selected and screened using simple sequence repeat markers DRRM-RF 3 -10 for Rf3 gene and RM6100 for Rf4 gene. One hundred and five plants possessing either or both of the genes were test crossed with CMS line COMS 23A. In 67 hybrids evaluated, the mean pollen fertility ranged from 97.3% (CB174R/Azucena 177-4-9) to 13.7% (CB174R/Azucena 13-2-4). The frequency of restorers was high (49.25%) followed by partial restorers (29.85%) and partial maintainers (20.90%). The selection efficiency for DRRM- RF3-10 and RM6100 markers were 75.75% and 54.54% respectively. Segregation for fertility restorer genes and pollen fertility among individual plants within a family was witnessed from molecular and phenotypic data. Based on phenotypic and marker information, it was concluded to advance 53.3% of plants to F5 generation to isolate stable restorer lines that can be exploited in future to produce highly heterotic three-line hybrids in rice.

Heterosis studies for grain yield and yield attributes in rice (Oryza sativa. L) hybrids

In the current study, 20 elite rice genotypes were crossed with four CMS lines- CMS 23A, CMS 59A, CMS 64A and JMS 13A in a line x tester mating design and assessed alongside standard controls (JKRH 3333 and MTU 1001) to determine the best heterotic combinations in terms of grain yield and yield component characters at the Rice Research Centre, Rajendranagar. Eight restorer lines were found in Kharif 2018–19 based on pollen and spikelet fertility (MTU 1153, RNR 26015, RNR 28355, JGL 25960, MTU 1010, IET 27253, RNR 26085 and JAYA). The resulting 32 hybrids of 8 restorer lines and their corresponding CMS lines are studied for combing ability and heterosis. The degree of heterosis varied between traits. Three of the 32 hybrids under study had considerable standard heterosis for grain yield viz., JMS 13A x MTU 1153, CMS 59A x RNR 26015 and CMS 64A x MTU 1153, which outperformed the best checks JKRH 3333 and MTU 1001. The best experimental hybrid, JMS 13A x MTU 1153, yielded the most grain yield per plant, 62.89g (Table 1). Further testing of these three experimental hybrids will take place in areas conducive to extensive commercialization. Keywords: Rice, Heterosis, Heterobeltiosis, Standard heterosis, Grain yield, Line x Tester

ASSESSMENT OF GENETIC VARIATION AND CHARACTER ASSOCIATION OF IRON, ZINC AND PROTEIN WITH AGRONOMIC TRAITS IN PEARL MILLET [Pennisetum Glaucum (L.) R. BR.

The present investigation consists of 98 genotypes (two CMS lines, 30 testers, 60 hybrids and six checks) studied to assess the magnitude of genetic variability and character association of iron, zinc and protein with other agronomic traits. Pearl millet grain has rich iron content, zinc content and protein and are essential for human nutrition. Estimation of genetic parameters viz., variability, heritability, genetic advance will provide the basis for selecting systematic breeding strategy to improve the genotypes with desirable traits. Moderate PCV and GCV values were observed for grain iron and protein content, high PCV and GCV values were observed for zinc content. Influence of environment on these traits is low because of the less difference between the estimates of GCV and PCV. High heritability coupled with high genetic advance as percent of mean was observed for zinc content. Strong correlation was observed between grain iron and zinc content and their weak correlations with other agronomic traits indicated there is a possibility to breed high grain Fe and Zinc contents simultaneously in different plant agronomic backgrounds might be suitable for different agro-climatic regions.

Studies on gene action and combining ability in rice (Oryza sativa. L) hybrids

The study was carried out to learn about the nature of gene action and combining ability in rice hybrids for grain yield and its component traits. For this, eight lines were crossed with four testers in an L x T mating design. The analysis of variance revealed significant variance for all the characters studied among the parents and hybrids. GCA variances were higher than SCA variances for most of the characters, indicating the predominance of additive gene action, while SCA variances were higher than GCA variances for number of productive tillers per plant, pollen fertility, hulling, milling, head rice recovery and grain yield per plant, indicating the predominance of non-additive gene action. The testers JMS 13A (3.91) and CMS 59A (2.46), as well as the lines MTU 1153 (9.52), RNR 28355 (3.26) and RNR 26085 (3.09), were the best combiners for grain yield and important yield components due to positive GCA effects. Based on SCA effects and corresponding mean performance for grain yield and its component traits, the hybrids CMS 59Ax RNR 26015, CMS 23A x JGL 25960, CMS 64Ax RNR 26015 and JMS 13Ax MTU 1153 were identified as the best specific combiners. These hybrids could be tested in multi-environment trials for commercial use. Keywords:  CMS lines, Gene action, Combining ability, GCA, SCA, Line x Tester &nbsp

Combining ability studies through Line × Tester design in sweet sorghum (Sorghum bicolor L.) for bioethanol production

In developing of sweet sorghum hybrids, non-additive genetic effects are important in phenotypic expression of the traits of interest. The present experiment was undertaken to assess the combining ability in sweet sorghum genotypes using 16 hybrids developed by crossing four CMS lines with four elite testers in a Line × Tester mating design. The hybrids were evaluated at three locations in  Andhra Pradesh in a Randomized Block Design with two replications during Rabi, 2018. Among the 13 quantitative characters studied the non-additive genetic variance was higher than the additive variance for all the traits except for days to 50 % flowering, days to maturity, plant height and ethanol yield. Among parents, ICSA 14029, ICSA 14030 (females) ICSV 15006 and GGUB-28 (males) were found to be  good general combiners for ethanol yield and its related traits with consistent performance across locations. ICSA 14029 × ICSV-15006 and ICSA 14033 × SEVS-08 were identified as superior crosses for the specific combining ability for high ethanol yield. Keywords: Sweet sorghum, Brix %, Bioethanol, gca, sca components

Molecular markers-based DNA fingerprinting coupled with morphological diversity analysis for prediction of heterotic grouping in sunflower (Helianthus annuus L.).

Cultivated sunflower holds a very narrow genetic base and the efficient utilization of available genetic diversity is very important for a successful breeding program. In the present study, 109 sunflower genotypes were assessed for diversity paneling through a combined approach of morphological and molecular markers analysis. Morphological parameters including days to flower initiation, days to flower completion, plant height, stem curvature, number of leaves per plant, leaf area, head diameter, hundred seed weight, and seed yield per plant were studied. Simple sequence repeats (40 DNA markers) were deployed for diversity profiling. Data were analyzed by both univariate and multivariate statistics. SD and coefficient of variation confirm the presence of significant amounts of genetic variation for all the morphological parameters. Cluster Analysis and Principal Component Analysis further confirm the presence of distinct grouping patterns in the studied material. Cluster analysis of both morphological and molecular analysis revealed that restorer lines tend to group separately from A, B, and open-pollinated lines. Further grouping, at the sub-cluster level, revealed six distinct sub-clusters in each of the two major clusters. In total, 12 genotypes, 6 CMS lines (CMS-HAP-12, CMS-HAP-54, CMS-HAP-56, CMS-HAP-99, CMS-HAP-111, and CMS-HAP-112) and 6 restorer lines (RHP-38, RHP-41, RHP-53, RHP-68, RHP-69, and RHP-71) could be used as potential parents for hybrid development. As genotypes of similar genetic backgrounds tend to group closer, it is deduced that one genotype with the highest seed yield per plant could be used for further hybrid breeding programs in sunflowers.

PRODUCTION OF SUNFLOWER HYBRIDS AND THEIR STUDY IN AUTUMN SEASON TO RECOGNIZE BEST YIELDING HYBRIDS

The research was in autumn season during the year (2019-20) at the research area of Rajawala farm, University of Agriculture, Faisalabad to study the best performing hybrids in Sunflower (Helianthus annuus L.) in autumn season. Experimental material was obtained from USDA and NARC. CMS lines and restorers were planted in the field and crossed in line × Tester. In next autumn season the crosses and their parents was evaluated with respect to their performance for yield and related traits in randomized complete block design (RCBD) using three replications. The data was taken on traits for days to germination, days to flower initiation, days to flower completion, days to flower maturity, plant height, internodal distance, number of leaves per plant, leaf area, number of whorls per flower, head diameter, 100 achene weight and achene weight per head. The data was first exposed to analysis of variance to find out the significant and non-significant differences among parents and crosses. Then combining ability and heterosis was calculated to find out the best general and specific combiners among the parents. 7-A, 23-A, 27-A, 80-R, 83-R and 96-R proved as good general combiners in both the seasons. 7-A x 86-R, 11-A x 83-R, 23-A x 81-R, 25-A x 80-R, 25-A x 94-R and 27-A x 80-R declared as best hybrids in terms of yield.

Screening of Rice Hybrids for Cold Tolerance at Seedling and Vegetative Stage under Low Temperatures

Low temperature is one of the most important abiotic constrains in rice cultivation. Rice plant growth and development are affected by cold stress from germination to reproductive stages. Development of 48 hybrids involving twelve restorer lines and four CMS lines by using line x tester mating design. Evaluation of 69 rice genotypes including checks based on leaf score (SES, IRRI, 2013), root length, shoot length, fresh weight, dry weight, and SPAD analysis at seedling stage as well as leaf score, number of tillers, seedling growth and establishment at vegetative stage. Results indicated that the hybrids IR72081 A x JGL 11118, IR72081 A x JGL 21071, IR80559A × JGL 29662, IR80559A × KNM 110, IR80559A × SN 470, IR58025A × JGL 29662 identified as cold tolerant genotypes. This study aimed to identify cold tolerant genotypes under low temperatures at seedling and vegetative stage. Screening for cold tolerance during early growth stages potentially be an effective way of assessing cold tolerance in breeding programmes.

Identification of a Candidate restorer-of-fertility Gene Rf3 Encoding a Pentatricopeptide Repeat Protein for the Cytoplasmic Male Sterility in Soybean.

The cytoplasmic male sterility/restorer-of-fertility (CMS/Rf) system plays a vital role in high-efficiency hybrid seed production in crops, including soybean (Glycine max (L.) Merr.). The markers linked to fertility restoration and the restorer-of-fertility (Rf) genes are essential because they can facilitate the breeding of new CMS lines and production of commercial hybrid soybean seeds. To date, several soybean Rf genes have been mapped to various genetic loci in diverse genetic populations. However, the mapping range of restorer genes remains narrow, with relatively limited practical applicability. Therefore, in the present study, F2 and F3 segregating populations derived from the CMS line JLCMS5A crossed with the restorer line JLR2 were developed and used for Rf3 gene fine mapping. Genetic investigation indicated that the restorer line JLR2 was controlled by a single dominant gene, Rf3. By integrating bulk-segregant analysis and next-generation sequencing, a 4 Mb region on chromosome 9 was identified, which was most likely the target region harboring the candidate gene responsible for fertility restoration. This region was further narrowed down to 86.44 Kb via fine mapping in F2 and F3 populations using SSR, InDel, and dCAPS markers. This region contained 10 putative genes (Glyma.09G171100–Glyma.09G172000). Finally, Glyma.09G171200, which encodes a mitochondria-targeted pentatricopeptide repeat protein, was proposed as the potential candidate for Rf3 using sequence alignment and expression analysis in restorer and CMS lines. Based on single-nucleotide polymorphisms in Glyma.09G171200, a CAPS marker co-segregated with Rf3 named CAPS1712 was developed. Our results will be fundamental in the assisted selection and creation of potent lines for the production and rapid selection of novel restorer lines.