Genetics of growth habit in horse gram [Macrotyloma uniflorum (Lam.) Verdc.]

Growth habit is a plant architectural trait in grain legumes with no exception of horse gram [Macrotyloma uniflorum (Lam.) Verdc.]. Determinacy and indeterminacy are the two types of growth habits reported in horse gram. Relative advantages of the two types of growth habit depend on the production systems to which cultivars are targeted. Dependable information on genetics of growth habit provide clues for adopting the most appropriate selection strategy to breed high yielding horse gram varieties with desired growth habit. Taking cues from the past studies, we hypothesize that growth habit in horse gram is controlled by two genes displaying inhibitory epistasis and indeterminacy is dominant over determinacy. To test this hypothesis, we monitored the inheritance of growth habit in F1, F2 and F3 generations derived from two crosses involving parents differing for growth habit. Contrary to our hypothesis, determinate growth habit of F1s of both the crosses suggested dominance of determinacy over indeterminacy. A good fit of observed segregation of F2 plants to that of the hypothesized segregation in the ratio of 13 determinate: 3 indeterminate plants, besides confirming dominance of determinacy, suggested classical digenic inhibitory epistatic control of growth habit. These results were further confirmed in F3 generation based on goodness of fit between observed numbers of plants segregating for determinacy and indeterminacy and those expected in the ratio of 49 determinate: 15 indeterminate plants. To the best of our knowledge, this is the first report on the inheritance of growth habit in horse gram.


Introduction
Horse gram [Macrotyloma uniflorum (Lam.) Verdc.] is one of the ancient, indigenous and underutilized grain legume crops widely grown in India (Verdcourt 1982). It is the fifth most important grain legume crop grown in India (Fuller and Murphy 2018). Its ability to grow in nutrient-poor soils and tolerate moisture stress makes it a crop of choice for resource-poor farmers in drought prone areas. It significantly contributes to protein requirement of a large number of resourcepoor farmers, especially those who depend on vegetarian diet. Besides these advantages, it serves as a valuable crop component to address present and future environmental challenges to agricultural production (Kahane et al. 2013). Despite its importance as a climate resilient grain legume crop, horse gram has received disproportionately little research compared to other legumes such as pigeonpea, chickpea and cowpea (Fuller and Murphy 2018).
Determinate and indeterminate are the two types of growth habits reported in horse gram. Unlike in other legumes such as common bean (Campos et al. 2010), chickpea (Sandhu et al. 2010) and dolichos bean (Keerthi et al. 2016) where the main meristem terminates in inflorescence in determinate genotypes, the main stem stops growing but will not terminates in inflorescence even when flowering starts in the lateral branches in horse gram (Ashwini et al. 2021). Almost all the currently used horse gram cultivars display indeterminate growth habit. Asynchronous flowering, and pod development and maturity driven by long overlapping vegetative and reproductive phases followed by shattering of first formed pods are attributed as important causes (among others) for poor grain productivity of cultivars with indeterminate growth habit (Huyghe 1998). On the other hand, greater synchronous flowering, pod development and maturity driven by shorter overlapping vegetative and reproductive phases are important features of genotypes with determinate growth habit (Kwak et al. 2012;Kato et al. 2019). Determinacy is a domesticationdriven plant architectural modification in grain legumes (Huyghe 1998). The compact growth habit of determinate genotypes facilitates high density planting that help maximize productivity (Kim et al. 1992). Synchronous pod maturity in determinate genotypes minimizes grain yield losses attributable to pod shattering. These features of determinate genotypes along with short crop growth and maturity period make them most ideal cultivar types suitable for mechanical harvesting and for multiple and intercropping production systems.
Wide acceptance and popularity of determinate cultivars in other legumes such as common bean (Mekbib 2003), chickpea (Sandhu et al. 2010;Hegde 2011), dolichos bean  and soybean (Kato et al. 2019) suggest that breeding and deployment of determinate cultivars should receive high priority in horse gram as well. However, the productivity of determinate cultivars is low due to their fewer branching and pod bearing abilities. Nevertheless, their compact growth habit enable high density planting which adequately compensates for fewer branches and pods and hence offers possibility to maximize their productivity (Ashwini et al. 2021). A precise knowledge on genetic basis of growth habit would provide useful clues (among others) for adopting the most appropriate selection and breeding strategies for developing determinate cultivars in horse gram. Taking cues from reported studies in related legumes such as pigeonpea (Waldia and Singh 1987) and chickpea (Sandhu et al. 2010;Hegde 2011), we hypothesize that the growth habit in horse gram may be controlled by two genes and indeterminacy is dominant over determinacy. The present study is aimed to test this hypothesis in horse gram.

Material and methods
The basic material consisted of two indeterminate cultivars namely Palem 1 and CRIDA-18-R and two determinate genotypes, namely HPKM-320 and IC 361290 (Table 1).
Development and characterization of experimental material for growth habit CRIDA-18-R and Palem 1 were used as male parents and HPKM-320 and IC 361290 as female parents to synthesize crosses. The tiny flowers were emasculated in female parents, the evening of the day before pollination on next day morning. The two crosses, namely HPKM-320 9 CRIDA-18-Rand IC 361290 9 Palem 1 were synthesized during 2018 post rainy season at the experimental plots of the Department of Genetics and Plant breeding (GPB), University of Agricultural Sciences, (UAS), Bangalore, India. A total of only eight and five well-filled F 1 seeds could be obtained from HPKM-320 9 CRIDA-18-R and IC 361290 9 Palem 1, respectively. The seeds of four parents, F 1 seeds were planted in 2019 summer season. The seeds of the four parents and all the F 1 seeds germinated and survived to maturity in 2019 rainy season. A total of 10 plants each of the four parents were maintained. The selfed pods from F 1 s of the two crosses were harvested, hand-threshed and sun-dried to obtain F 2 seeds. F 2 plants from these two crosses were raised in 2019 post rainy season. A total of 259 and 245 F 2 plants from HPKM-320 9 CRIDA-18-R and IC 361290 9 Palem 1, respectively survived to maturity. Selfed pods from each F 2 plants were manually harvested, hand-threshed and seeds were sun-dried for use in raising F 3 population during 2020 rainy season.
As a result of mortality of several plants at seedling stage due to natural infection by yellow mosaic virus, fewer than expected numbers of F 3 families survived to maturity. However, total number of plants that survived to maturity in F 3 generations of both the crosses (389 in HPKM-320 9 CRIDA-18-R and 211 in IC 361290 9 Palem 1) were still sufficiently large enough to get reliable data on growth habit. The recommended production package was practiced to raise parents, F 1 , F 2 and F 3 generations.

Data recording and statistical analysis
Each plant of the parents, F 2 and F 3 plants of the two crosses were inspected and their growth habit was recorded at maximum pod formation stage. Two distinct growth habits could be observed in F 2 and F 3 generations of both crosses. All the plants bearing elongated flowering branches that terminated in vegetative bud similar to those in indeterminate parents were classified as indeterminate (ID); and plants bearing short primary and secondary branches with their apical buds developing into a flower bud or fully opened flower similar to those in determinate parents as determinate (D) (Hegde 2011). The number of plants displaying determinate and indeterminate growth habits were counted. Based on hypothesized inheritance pattern (digenic inhibitory epistasis) (Sandhu et al. 2010;Hegde 2011), goodness of fit between observed and expected segregation ratio of 13 determinate: 3 indeterminate F 2 plants and 49 determinate: 15 indeterminate F 3 plants were examined using v 2 test. The analysis was implemented using ''Data analysis'' option of Microsoft Excel software. Non-significance of v 2 test was considered as an evidence for digenic inhibitory epistatic genetic control of growth habit and dominance of determinacy over indeterminacy.

Results and discussion
Contrary to our hypothesis, the F 1 's of both the crosses displayed determinate growth habit (Fig. 1). Hence, we suspected that the F 1 's are not true crosses. However, appearance of both determinate and indeterminate plants in F 2 generation not only confirmed that the F 1 's were true crosses but also confirmed the dominance of determinacy, contrary to our hypothesis. Further, a greater frequency of determinate plants and a good fit of observed numbers of determinate and indeterminate plants to those expected based on hypothetical segregation ratio of 13 determinate to 3 indeterminate plants in F 2 generation (Table 2) suggested that growth habit in horse gram is controlled by  (Table 3). Thus, our results based on F 2 and F 3 segregation indicated that the growth habit in horse gram is controlled by two genes that exhibit inhibitory epistasis and determinacy is dominant over indeterminacy. The dominance of determinacy over indeterminacy has also been reported in spaghetti squash (Cucurbita pepo) (Edelstein et al. 1989) and common bean (Phaseolus vulgaris) (Campos et al. 2010). Waldia and Singh (1987) in pigeonpea, and van Rheenen et al. (1994) and Sandhu et al. (2010) in chickpea have also suggested digenic inhibitory epistatic control of growth habit. However, they reported dominance of indeterminacy over determinacy. We believe that dominance of determinacy in horse gram is not surprising considering that dominance of any trait's alternate alleles (Kearsey and Pooni 1996) depends on the level of organization of phenotypes controlled by them (Mike 2008), and that the architecture of determinate phenotype in horse gram is different from that reported in common bean (Koinange et al. 1996;Campos et al. 2010), chickpea (van Rheenen et al. 1994Sandhu et al. 2010;Hegde 2011) and in dolichos bean (Keerthi et al. 2014(Keerthi et al. , 2016. It is likely that different genotypes may harbor different genes controlling determinacy (Edelstein et al. 1989). It is also possible that dominance of alternate alleles at different genes may vary with genotype. The arguments on these possibilities suggest that determinacy in horse gram might also inherit as a dominant phenotype, if genotypes different from those used in the present investigation will be used to decipher inheritance of determinacy. The reported results on dominance of both indeterminacy (Koinange et al. 1996) and determinacy (Campos et al. 2010) using different sources of determinacy in common bean lends adequate support to our results and arguments thereof.
We designate the two genes controlling growth habit in horse gram as 'D' [with 'D' (dominant allele) and 'd' (recessive allele)] and 'ID'[with 'ID' (dominant allele) and 'id' (recessive allele)]; while gene 'D' controls determinacy, 'ID' controls indeterminacy. The gene 'D' either in dominant or recessive state in the absence of dominant allele 'ID' produces determinate growth habit. The gene 'ID' in dominant state inhibits 'D' only in its recessive state. Based on this mode of action of 'D' and 'ID' genes controlling the inheritance of growth habit, we have proposed most probable genotypes of parental, F 1 and F 2 populations (Table 4). To the best of our knowledge, this is the first report on the inheritance of growth habit in horse gram.

Implications in breeding horse gram
If the objective is to develop determinate cultivars in horse gram, determinate plant needs to be selected right in F 2 generation (to fix the determinacy), followed by selection for high grain yield potential

Declarations
Conflict of interest Authors of this manuscript declare that we have no conflict of interest.
Consent for publication All the authors have provided the consent for publication.
Ethical approval This manuscript does not contain any studies with human participants or animals performed by any of the authors.