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Damayanti, Farida (2010) Comparative studies on cultivated and wild accessions of Vigna vexillata (L.) A. Rich. Masters (Research) thesis, James Cook University.

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5.0 STUDIES OF POLLEN GRAIN VIABILITY AND CHROMOSOME NUMBER IN J': VEXILLATA 5.1 Introduction As outlined in Chapters 3 and 4, respectively, 19 accessions have been analysed for a number of qualitative and quantitative traits, and hybrids were generated using 17 of these accessions. Genetic barriers were apparent in the hybrids between cultivated Bali and wild accessions. This was evident in the following outcomes: (i) non viable hybrid (F t) seeds (for instance,CPI 16683 x Jim lA, cpr 60458 x Tab 2 and Jim 2 x CPI 69030); (ii) the cessation ofgrowth at c. 18 days after germination (Jim 1A x ACC 351 and Tab 3 x ACC 351); and (iii) hybrid sterility in the crosses of ACC 390 x Jim 1A and ACC 390 x Jim 2. In the last type of genetic barrier, the hybrids failed to set pods even when tripping was attempted to obtain F2 seed. As described in Chapter 4, backcrosses were carried out on ACC 390 x Jim 1A and ACC 390 x Jim 2 hybrid combinations. There was a low percentage ofpod set and a low number ofseeds set per pod.

Genetic breakdown may be attributed to a variety of mechanisms, depending on the genus and species examined (Mutschler and Liedl 1994). For instance, irregular chromosomal pairing and subsequent rearrangement during meiosis often causes sterility in the hybrid between two distantly related species (Ladizinsky 1998; Rieseberg and Carney 1998). However, genetic breakdown also has been repOlted in intraspecific hybridisation. Poor growth performance in the hybrids and hybrid breakdown have been reported in intraspecific hybridisation in rice. The breakdown was attributed to the interaction of two recessive genes (Li et al. 1997; Kubo and Yoshimura 2002; Yamamoto et al. 2007). Unfortunately, so far it has been difficult to distinguish unambiguously between chromosomal and genic effects (Rieseberg and Carney 1998), except when the hybridisation experiment has been supported by DNA marker analysis, such as Restriction Fragment Length Polymorphism (RFLP), as has been done with rice (Li et al. 1997; Kubo and Yoshimura 2002).

Yamamoto et al. (2007) suggested that clarifying the mechanisms behind reproductive barriers such as hybrid breakdown is important not only to understand biological speciation but also to facilitate removal ofthe battier for crop breeding. Therefore, the purpose ofthis experiment was to investigate the cytological basis for the genetic breakdown which was observed between some cultivated and wild type accessions of V vexillata.

80 5.2 Material and methods 5.2.1 Germplasm The cytological studies in Experiment 3 involved: (i) analysis ofpollen viability and (ii) mitotic analysis of root tip squashes, to detennine somatic chromosome number. Selected cultivated and wild V vexillata accessions and their hybrids, produced as explained in Chapter 4, were investigated in these studies (Table 5.1).

Table 5.1 The two wild, two cultivated and five hybrid accessions of V. vexillata used in cytological studies. Hybrids are designated as male x female parent. Observations were made of 1. pollen viability and 2. mitosis.

Germplasm type Genotype Observation

Wild ACC 390 1,2 Wild ACC 351 2 Cultivated Jim lA 1,2 Cultivated Jim 2 1,2 Hybrid ACC 390 x Jim lA 1,2 Hybrid ACC 390 x Jim 2 1,2 Hybrid Tab lA x ACC 351 2 Hybrid Tab 3 x ACC 351 2 Hybrid ACC 351 x Tab 3 2

5.2.2 Cytological procedures 5.2.2.1 Pollen viability analysis a. Pilot experiment Based on a pilot experiment, Alexander's staining procedure was chosen to test pollen viability in parental accessions and hybrids. In the pilot experiment, pollen grains sampled from ACC 390 were stained using either Alexander's stain or aceto-carmine dye. Based on ANOVA analysis that was performed using SPSS 16.0 Graduate Student Version software (SPSS, Inc), preliminary data showed that the percentages of pollen viability detected by aceto-carmine dye were not significantly different than was evident using Alexander's stain (Table 5.2). However, Alexander's stain was considered to be a more reliable indicator of pollen viability because it discriminated more clearly between viable and non viable pollen grains. When Alexander's stain was used, viable pollen grains stained dark purple-red, while non viable pollen grains stained pale blue-green. When aceto-carmine was used, the contrast was red pink for viable grains and light pink for non viable grains. Dafni (1992) cautioned against the use of aceto­ cannine, pointing out that pollen stainability may not be a valid index of pollen viability. Alexander (1969) pointed out that dyes such as aceto-carmine and cotton blue in lactopheno1 do not stain the aborted pollen, whilst Alexander's stain does.

81 Table 5.2 Comparison of aceto-carmine and Alexander's stain in determining pollen viability in V. vexillata (ACC 390). In each case, the percentage of viable pollen shown is based on 10 anthers excised from a single flower.

Sub samples Aceto-carmine stain Sub samples Alexander's stain 95.7 % 4 95.3 % 2 96.8 % 5 96.7 % 3 97.4 % 6 96.0 % Range 95.7 %- 97.4 % Range 95.3 %- 96.0 % b. Analysis of pollen viability in five ofthe nine accessions listed in Table 5.1 As indicated in Table 5.1, pollen viability was analysed in accessions ACC 390, Jim lA, Jim 2 and the hybrids ofACC 390 x Jim lA and ACC 390 x Jim 2. Ten flowers were sampled, except for ACC 390 in which the case only five flowers were available at the time of sampling. Samples were collected between 0700 and 0800 hours between February 25 and March 15, 2008. Pollen grains were collected from the ten anthers in each ofthe flowers collected.

Analyses of pollen viability were can-ied out as follows: all pollen grains from ten anthers in each flower were transferred onto a clean slide, stained with a drop ofAlexander's stain solution and then covered with cover slip for c. 10 minutes prior to observation. Viable and nonviable pollen grains were observed and counted under a high power (H.P.) microscope at x40 magnification in different fields of view (Olympus System Microscope Model BHS, Olympus Optical Co., LTD, Tokyo, Japan) and photographed using an Olympus DP12 digital camera. Based on the numbers of dark-purple red and blue-green pollen grains observed, the percentage of viable and non viable pollen was detennined and the data was analysed using SPSS 16.0 Graduate Student Version software (SPSS, Inc) to determine differences in pollen viability among parental accessions and their hybrids.

5.2.2.2 Mitotic analysis In order to determine the chromosome number in each parental accession and its hybrids, root tips were used in mitotic studies. The root tipping method was modified from the technique used in Vigna lanceolata by Bull (1987). Prior to genninating them, seeds were scarified than allowed to germinate in petri dishes lined with filter paper that had been moistened with distilled water. Both the radicle and lateral roots were used in mitotic analyses. When the radicle was c. 10 mm long, the terminal c. 7 mm was excised for root tipping. Lateral roots proliferated from the remaining 3 lllill ofradicle. They too, were excised and used in root tipping when they were c. 10 mm long.

82 Both radicle and lateral roots were pre-treated in 0.002 M 8-hydroxyquinoline at 20°C for 4 hours, fixed in 45 percent acetic acid for one minute, and macerated in a mixture of IN acid mixture had been pre-warmed to 60°C for five minutes before use. Roots were then stained with I percent aceto-orcein for between four to eight hours at room temperature, then the terminal I mm of each root tip was cut, placed onto a clean slide, a drop of aceto-orcein was added, and the tip was cut into smaller pieces, and a coverslip was applied. The slide was then placed in folded blotting paper, held steady between two fingers and tapped gently with a soft pencil to further break up the root tip. It was followed by squashing, which was done by pressing down very firmly on the coverslip with the thumb. Chromosomes were observed and counted under a high power (H.P) microscope (Olympus System Microscope Model BHS, Olympus Optical Co., LTD, Tokyo, Japan) at xIOO magnification with oil immersion and photographed using a digital camera (Olympus DPI2).

Chromosome number was determined for a minimum 10 cells for each accession and the hybrids whenever possible. However, no cell count was obtained in one hybrid because of a limited number ofavailable seed (one), which failed to germinate.

5.3 Results and discussion 5.3.1 Pollen viability analysis Generally, viable pollen can be easily distinguished fi:om non viable pollen based on colour and size differences as presented in Plate 5.1. Viable pollen grains stained dark purple-red whilst non viable pollen stained pale blue-green. Moreover, viable pollen was larger (c. 80 f.Ill1.) than non viable pollen (c. 50 /-Un) (Plate 5.1). Kelly et al. (2002) reported positive correlations between pollen size and pollen viability in Mimulus guttatus and Collinsia verna. In spite ofthe correlations between those two traits, Kelly et al. (2002) pointed out that determination of pollen viability could not be based solely on the pollen size, because some non-genic factors affected pollen size and also pollen viability, e.g. temperature and humidity.

Based on the ANOVA, there were large and statistically significant (P ::; 0.0 I) differences among genotypes (ACC 390, Jim lA, Jim 2, ACC 390 x Jim lA, and ACC 390 x Jim 2) and also between germplasm types (wild parent, cultivated parent, hybrid) for the percentages of viable pollen. This was illustrated by the LSD test, which showed that there were significant differences between the wild parent ACC 390 and the two cultivated Bali parents Jim lA and Jim 2. ACC 390, Jim lA and Jim 2 were also significantly different from the hybrids (Table

83 5.3), but Jim IA and Jim 2 were not significantly different from each other. Nor were the two hybrids ACC 390 x Jim IA and ACC 390 x Jim 2 significantly different from each other. ... e. .'....­ ..

• .. " • V· ~ ~ 0 = = ~ll!Uil ""'= •• \ • • •• • • ~ •• • • • • • 4 •• •• • •• •• • [!]

Plate 5.1 Pollen grains of V. vexil/ala were stained using Alexander's stain and are shown at x40 and xlO magnifications. Viable pollen stained dark purple-red, non viable pollen stained pale blue­ green. a and b were pollen grains of ACC 390 (wild parent). c and d were pollen grains of ACC 390 x Jim IA (hybrid). e and fwere pollen grains of Jim IA (cultivated Bali parent). All pollen grains in hybrid shown were non viable (c and d).

The wild parent (ACC 390) had the highest percentage of viable pollen (95.94 percent), followed by the cultivated Bali parents with 76.15 percent in Jim IA and 67.79 percent in Jim 2.

84 In contrast, only 4.25 percent and 4.44 percent ofviable pollen was observed in ACC 390 x Jim lA and ACC 390 x Jim 2, respectively (Table 5.3). Low pollen viability (27.8 percent) in hybrids also has been icportcd b)~ GomathinayTagam et al. (1998) who investigated inter-specific hybridisation between V vexillata and V unguiculata. In that study, the aceto carmine squash technique was used to identify viable and non viable pollen grains.

Table 5.3 Mean percentages of viable pollen observed from ten anthers in each flower of the wild parent (ACC 390), cultivated Bali parents (Jim lA and Jim 2) and their hybrids. Means within a column followed by the same letter are not significantly different (P > 0.05).

Germplasm Genotype Viable pollen (%) S.E. Range (%) Wild parent ACC 390 95.94 a 0.216 95.33 - 96.67 Cultivated Bali parents Jim 1A 76.15 b 6.623 20.48 - 94.91 Jim 2 67.79 b 6.776 30.48 - 93.15 Hybrids ACC 390 x Jim 1A 4.25 c 0.452 1.22 - 6.10 ACC 390 x Jim 2 4.44c 0.448 2.57 - 6.50

Compared to the wild parent and the hybrids, larger variation in the percentage of viable pollen was observed in the cultivated Bali parents, which was indicated by larger values of standard en-or (S.E.) (Table 5.3). Apart from the effect of genotypic expression on the percentage of viable pollen, pollen viability was possibly also influenced by other factors such as prevailing temperature during floral development. High temperature, particularly high night temperature during pre-anthesis (>20 0c), which occurred during flower sampling, has been repOlied to reduce pollen viability in legumes, e.g. in cowpea (Ahmed et at. 1992), in groundnut (prasad et al. 1999) and in common bean (porch and Jahn 2001). Level of sensitivity to high temperature was different among species, or even within species (Ehlers and Hall 1998; Rebetzke and Lawn 2006b). However, for the data presented in Table 5.3, it is not possible to ascribe different viabilities to different temperature regimes, because oflimited number offlowers sampled.

Based on ANOVA, large and statistically significant (P $; 0.01) differences among genotypes and among germplasm types (wild parent, cultivated parent, hybrid) were observed for the number ofpollen grains in each sampled flower. Table 5.4 shows that differences in the number of pollen grains in each sampled flower were most apparent when comparing the generations. As was the case in pollen viability analysis, there were no significant differences among cultivated parents (Jim lA and Jim 2) and also among the hybrids (ACC 390 x Jim lA and ACC 390 x Jim 2) (P > 0.05) in the number ofpollen grains in each sampled flower (Table 5.4).

85 Table 5.4 Mean number of pollen grains counted from ten anthers in each flower sampled from the wild parent (ACC 390), cultivated Bali parents (Jim lA and Jim 2) and their hybrids. Means within a column followed by the same letter are not significantly different (P > 0.05).

Number of pollen Germp1asm Genotype S.E. Range grains Wild parent ACC390 1,699.0 a 128.6 1,214 - 1,904 Cultivated Bali parents Jim lA 1,051.3 b 249.9 166 - 2,434 Jim 2 945.3 b 207.6 180 - 1,730 Hybrids ACC 390 x Jim 1A 241.7 c 23.0 162 - 353 ACC 390 x Jim 2 336.6 c 39.2 123 - 542

The wild parent (ACe 390) produced the highest number of pollen grains in each sampled flower (mean 1,699) compared with the four other accessions studied. Cultivated Bali parents produced about 1000 pollen grains per flower while hybrids produced 242 - 337 pollen grains per flower (Table 5.4), As had been observed in the pollen viability analysis, there was a wider range of viability in the cultivated Bali parents, than in the wild parent (Table 5.3) and this is consistent with the wider range in number ofpollen grains number in each sampled flower (166­ 2434 and 180-1730), compared to wild parent and the hybrids (Table 5.4). Possibly, this may be due to the effect of high temperature on pollen production. The cultivated Bali accessions are adapted to temperatures ofabout 36°C in Bali, and in the shade house from which samples were collected in Townsville, daily minima and maxima per week exceeded 24.4°C and 40.3°C. Such high temperature may have adversely affected both pollen production and pollen viability, There was highly significant conelation (P ~ 0,01) between number of viable pollen grains and number of pollen grains per flower in this experiment (r = 0.76), It suggests that genetic breakdown is not only influencing pollen viability but also pollen production in the hybrids.

5.3.2 Mitotic analysis The data showing chromosome number and the number of cells counted is presented in Table 5.5. In most accessions, chromosome number was successfully determined based on at least 10 cells for each genotype, even though there were limited numbers of available seed particularly in the hybrids. Low germination rate and the failure to produce lateral roots was a problem in some genotypes, i,e. Tab 1A x ACC 351 and Tab 3 x ACC 351. In the case ofACC 351 x Tab 3, no cell count was obtained because the one hybrid seed failed to germinate, In order to obtain reliable counts of20 somatic cells, e.g. in ACC 390 (Table 5,5), it was necessary to examine 10 - 15 root tip preparations. Parida et al. (1990) pointed out that small chromosome size was one ofthe difficulties ofdoing cytogenetic investigations in Vigna. As observed in Plate 5.2b, based on extrapolation from the scale bar, the chromosome size of Jim 1A was about 1.5 J..I11l' Small chromosome size (1.76 pm - 3,29 J..I11l) also has been reported in V. lanceolata by Bull (1987).

86 Table 5.5 Number of cells counted and number of chromosomes observed at mitosis in two wild, two cultivated and four hybrid accessions of V. vexillata. Hybrids are designated as male x female parent.

Number ofchromosomes Germplasm Genotype Number of counted cells observed

Wild parents ACC 390 20 22 ACC 351 10 22 Cultivated parents Jim lA 18 22 Jim 2 10 22 Hybrids ACC 390 x Jim lA 15 22 ACC 390 x Jim 2 11 22 Tab lA x ACC 351 12 22 Tab 3 x ACC 351 10 22

The analysis ofsomatic cells of V vexillata genotypes indicated that all the genotypes have 2n = 22 chromosomes (Table 5.5, Plate 5.2). This chromosome number has been also reported in V vexillata by Adetula et al. (2005). Lackey (1980) suggested x = 10 and x = 11 as the basic chromosome numbers found in the genus Vigna. Other basic chromosome numbers also have been reported in the genus Vigna, e.g. x = 9 in V candida (Forni-Martins 1986) and x = 22 in V glabrescens (Lawn 1995). The chromosome numbers inserted in Plate 5.2 are regarded as valid because counting was done while adjusting focus on the microscope, but the photographic plate is one dimensional.

The fact that all the accessions studied had a somatic chromosome number of 22 indicates that differences in chromosome number could be eliminated as the cause of genetic breakdown leading to hybrid inviability and sterility. This does not negate the possibility that structural differences in chromosomes of the different accessions may be a contributory factor to genetic breakdown.

To ascertain the nature of such differences requires detailed studies of chromosome pairing behaviour at meiosis. This would pennit the detection of inversions, deficiencies, duplications and translocations all of which represent structural alterations in chromosomes and which impact adversely on the production of viable gametes. Such detailed cytological analysis was beyond the time frame available for this research but is recommended for the future.

87 ~:;~~i-;~~;.,~;.,~',-.~.1.'!·::.:~.',:~·.;.:".'.~:.' """~"""."'"'~" ~.:I.'.~.~~~~· rL':.~,:_'· :~:.'...... •,•. -,.•..•. •• .•...•..:.•'.,'.'•. .. . ':,':', ,,,v,"j. :.-;

~~~:{ "<, ,.'

Plate 5.2 Mitotic cells with 2n = 22 chromosomes observed in V. vexillata (a. ACC 390 and b. Jim lA). Scale bar was 6 JIm.

88 5.4 Summary and conclusions Alexander's stain distinguished the viable and non viable pollen in V. vexillata, because of the dear colour discrimination between them. Low poilen viability and a low number of poilen grains were observed in the hybrids between the cultivated Bali and wild accessions of V vexillata compared to their parental accessions. Even though the validity of pollen viability analysis has been subject of discussion (Dafni and Firrnage 2000), it does yield comparative data and the results presented in this thesis suggest that genetic breakdown which occurred in the hybrids between the cultivated Bali and the wild accessions of V vexillata were influenced by pollen development which was expressed in terms of both impaired pollen production and reduced pollen viability.

Hybrids resulting from crosses between parents with different chromosome numbers would produce gametes that are unbalanced in term of chromosome number and this could lead to genetic breakdown in those hybrids. Based on mitotic analysis, there was no difference in the number ofchromosomes in the two cultivated Bali and two wild accessions used in the analysis. However, genetic breakdown may occur in the hybrid of two species with the same number of chromosomes, as has been reported in lice (Li et al. 1997; Kubo and Yoshimura 2002; Yamamoto et al. 2007).

The data from pollen viability and mitotic analyses in the absence of meiotic analysis did not permit specification ofthe mechanism ofgenetic breakdown between the cultivated Bali and the wild accessions. However, these two analyses: (i) provided the initial information that very low pollen viability occurring as a result of genetic breakdown may have caused partial sterility in ACC 390 x Jim lA and ACC 390 x Jim 2 hybrids; and (ii) confirmed that the cultivated Bali (Jim lA and Jim 2) and wild (ACC 390 and ACC 351) accessions have the same chromosome number, which implies that chromosome number was not the causal factor of the genetic breakdown.

Meiotic analysis is necessalY to further investigate the genetic breakdown mechanism which occurred in the hybridisation ofthe cultivated Bali and the wild accessions of V vexillata. In the future, chromosome banding studies and the use ofmolecular markers could also be informative in attempts to elucidate the mechanism ofgenetic breakdown in V. vexillata.

89 6.0 INHERITANCE OF SELECTED TRAITS IN CULTIVATED AND WILD ACCESSIONS OF VIGNA VEXILLATA 6.1 Introduction One of the basic strategies in plant breeding is searching out genes that encode useful traits from cultivated species and their close relatives (SIeper and Poehlman 2006). Equally important is the need to understand how deleterious or unwanted traits are inherited. The development of an understanding of inheritance pattern and segregation ratios is thus an important step in crop improvement. In Vigna, inheritance of agronomic traits in mungbean (Sriphadet et al. 2007), and inheritance oftime to flowering, flower colour, pod colour, seed colour, leafmark, and seed dehiscence in cowpea (Aliboh et at. 1996; Sangwan and Lodhi 1998; Ishiyaku and Singh 2004; Othman et at. 2006; Adeyanju et at. 2007; Mustapha 2008; Mustapha and Singh 2008) have been reported. The inheritance in Vigna of a wide range of traits was documented by Fery (1980).

In contrast to Vigna generally, relatively few inheritance studies have been conducted in V. vexillata. James and Lawn (1991) evaluated the inheritance of qualitative and quantitative traits in hybrid populations generated by crossing wild genotypes ofAflican and Australian origin. In a more targetted study, Ogundiwin et al. (2002) evaluated populations of V. vexillata as a source of resistance to cowpea mottle virus, as part of a cowpea improvement program. Given the scarcity of inheritance studies in V. vexillata, more information is needed to provide a basis for future plant breeding efforts to accelerate varietal development. The purpose of the experiments repOlied below was to document the inheritance of selected traits using hybrids between diverse genotypes. Emphasis was placed on the inheritance of cultivated vs. wild traits in segregating hybrid populations from crosses between cultivated and wild type accessions.

6.2 Material and methods 6.2.1 Germplasm

Population development, which included the development ofFt and F2 hybrid populations, and backcrosses (Be) between F I plants and the two respective parents (BCP 1 and BCP2), was carried out as described in Chapter 4. As detailed previously, in the case ofthe cultivated Bali x wild and cultivated Bali x var. macrosperma crosses, most hybridisations failed to result in pods and lor the formation of viable seed (refer Table 4.2b). In two instances, involving crosses between the wild Australian genotype ACe 390 and the cultivated Bali genotypes Jim lA and

Jim 2, viable but se1f-infe11ile F 1 plants were obtained. Because ofthis fertility barrier, no selfed

F2 generation seeds were able to be obtained for these crosses. However, there was some limited success in obtaining backcross seeds on the F I plant using pollen from the two parental lines.

90 In contrast, no hybrid breakdown was observed in the val'. macrosperma x wild hybrids, using both African and Australian wild genotypes. The hybrid between val'. macrosperma CPI 69030 and the wild Australian genotype ACC 390 was chosen for the inheritance study because in Experiment 1, these two parents exhibited extremes of diversity in morphological attributes. The var. macrosperma parent was characteristically robust, with thick stems, large ovate leaflets, non-dehiscent pods and large uniformly coloured seeds, while ACC 390 was typically gracile, with thin stems, small narrow leaflets, strongly dehiscent pods and small dark coloured seeds. As discussed in Chapter 3, there were many morphological similarities between var. macrosperma and the cultivated accessions from Bali. In the absence ofany hybrid breakdown, parental, F I> BCPI> BCP2, and F2 populations were available for study using the var. macrosperma x wild combination (Table 4.2b).

6.2.2 Cultural details The evaluations of the cultivated Bali x wild backcross populations were calTied out between September 11, 2008 and December 20, 2008, in the shade house facilities of the School of Marine and Tropical Biology at James Cook University in Townsville, Queensland, Australia. The evaluation of the var. macrosperma x wild hybrid population was conducted between September 17, 2008 and JanualY 11, 2009, in the Genetic Garden facilities of the School of Marine and Tropical Biology at James Cook University in Townsville, Queensland, Australia. As observed in Experiment 1 (see Chapter 3), the Bali accessions exhibited sensitivity to long photoperiods. Therefore, in order to obtain the information about inheritance photoperiod sensitivity in backcross plants in the Bali x wild backcross populations, early summer was chosen for the experiment. Meanwhile, the evaluation of phenotypic expression of selected traits was carried out between July 15, 2008 and November 15, 2008 for the hybrid of CPI 69030 x CPI 29141 and its parent accessions and between September 25, 2008 and December 20,2008 for the hybrid ofCPI 69030 x CPI 60458 and its parent accessions.

For all evaluations, the plants were grown in spaced cylindrical PVC pots which were located on wire mesh benches c. 1 m above the ground. Individual pots were 200 mm in diameter and 200 mm depth and were painted silver to reflect radiation and so reduce the temperature inside the pots. The pots were filled with equal volumes ofvermiculite and watered prior to sowing, as outlined previously in Section 3.2.2. Prior to sowing, the seeds were scarified and germinated as described previously in Section 3.2.2. Germinating seeds were sown two per pot for the Bali x wild backcross populations and one per pot for the var. macrosperma x wild hybrid population, at a depth of c. 1.5 em, when the radicle root was c. 5 mm long. After plants were established, a 1.2 m bamboo stake was placed vertically in the pot to provide support as the plant needed it. An application of 30 g Osmocote Plus® controlled-release fertiliser was given to each pot at

91 sowing time to provide sufficient nutrients for vigorous growth. Plants were watered daily by hand. To ensure plants were not stressed between waterings, additional water was provided using a water-filled saucer under each pot.

As outlined in Chapters 3 and 4, insecticides and fungicide were applied whenever deemed necessary during the plant growth to avoid damage from insects and diseases. For the hybrid population located in the open environment of the Genetic Garden, the insecticide Imidacloprid (0.125 g.r') was applied to control the pod-sucking bug (Riptortus serripes), the brown bean bug (Melanacanthus scutellaris), and the green vegetable bug (Nezara viridula). In the shade house, applications of wettable sulphur (2.75 g,tl) were given to control powdely mildew (Podosphaera xanthii) and spider mite (Tetranychus urticae) that mostly infested the plants in the summer when the temperatures and humidity inside the shade house were very high. In addition, the floor of shade house was hosed regularly, to moisten the environment in order to reduce the infestation of spider mite, and the door of the shade house was left open from October 10,2008 until the end ofthe experiment to reduce the inside temperature.

Pods on several of the plants located in the Genetic Garden suffered damage from rainbow lorikeet parrots (Trichoglossus haematodes), with >10% of pods affected in the worst instances. The birds caused damage when about 50% of the plant population had set pods and used their beaks to remove the developing seeds from pods that were about to mature in c. 3-4 days. Most of the plants which were damaged by the birds were observed to have relatively larger seeds. Efforts applied to overcome the problem included hanging shiny computer compact disks around the plants to distract the birds and manually scaring them away by staying at the Genetic Garden during their feeding time from 6 am to 9 am and 4 pm to 6 pm each day.

6.2.3 Experimental design, data collection and statistical analysis

For each ofthe two Bali x wild hybrids, six plants ofeach parent, six F1 plants and the available backcross plants were grown in a completely randomised design. All pots were moved to a new location two times during the experiment to reduce possible local spatial effects. The numbers of backcross plants differed for each parent in each combination, reflecting the differences in hybridisation success during population development as described in Chapter 4. For ACC 390 x

Jim lA, there were nine backcross plants to ACC 390 (BCP 1) and 12 backcross plants to Jim 1A

(BCP2), while for ACC 390 x Jim 2, there were there were 11 backcross plants to ACC 390

(BCP,) and 43 backcross plants to Jim 2 (BCP2).

For the var. macrosperma x wild combination, the populations evaluated comprised 10 plants of each parent, six F. plants, 25 BCP. plants (wild parent), 24 BCP2 plants (var. macrosperma

92 parent) and 99 F2 plants. The population was grown in a completely randomised design. As for the Bali x wild populations, all pots were moved to a new location two times during the experiment to reduce possible spatial effects (e.g. exposure to wind or shading from nearby trees). Because of a shortage of available FI flowers when the backcross hybridisations were being made, only eight BCP J seeds were available for sowing with the other plants on September 17, 2008. An additional 17 BCP. seeds, from a later-set pod, were sown on October 25, 2008. The qualitative data measured on the eight early and the 17 late sown plants were combined in subsequent analyses. However, it was considered that for most of the quantitative traits, and particularly the phenological traits, the environmental differences due to the different sowing times would be too great.

In the two Bali x wild populations, differences were observed in the sizes ofthe backcross seeds

(i.e. BCP t and BCP2 generations) despite the fact that in all cases, the Bali accession was the maternal parent. In order to explore whether these differences may have had subsequent effects, the backcross seeds were separated into four size classes based on a visual assessment of the seed. The average size of the different classes, smallest to largest, were 0.6, 1.1, 1.6 and 2.3 g per 100 seeds for the BCP.. whilst the 100 seed weight for the BCP2 classes were 0.7, 1.4,2.2, and 2.9 g, respectively. A somewhat wider range of variation was observed in the size of seeds obtained from the F I plants in the var. macrosperma x wild population, and the seeds were classified visually into five different size classes. The average weight of 100 seeds for seed classes 1 to 5 were 1.9,2.7,3.3,3.9, and 4.4 g, respectively.

Several qualitative (Table 6.1) and quantitative (Table 6.2) attributes, based in part on the descriptors used by James and Lawn (1991) and Grant et al. (2003), were observed and measured for each single plant within both the Bali x wild and var. macrosperma x wild populations. All the qualitative and quantitative descriptors were scored and measured as outlined in Section 3.2, with the exception that data on flowering, and pod and seed traits were not obtained from many of the Bali cultivated x wild plants. Firstly, due to their extreme sensitivity to photoperiod, all the Bali plants and some ofthe backcross plants to the Bali parent had not produced flowers by the time the experiment was necessarily ended (late December 2008). Given the experience with the Bali accessions in Experiment 1 (Chapter 3), it is likely that these plants would not have flowered until the shorter days of autumn (March - April). Secondly, the F. plants and most ofthe backcross plants that flowered failed to set pods.

93 Table 6.1 Qualitative traits obsenTed in the inheritance studies with cultivated Bali x wild and with var. macrosperma x wild hybrid populations of V. vexillata.

Trait Definition Leaflet shape 1. Linear lanceolate leaflets with obtuse bases 2. Ovate leaflets with cuneate bases Leaflet width 1. Narrow; mean length/width ratio> 2.5 2. Broad; mean length/width ratio « 2.5 Leaflet pubescence 1. Short « 0.5 mm) 2. Long (> 0.5 mm) Stem pigmentation* 1. Full anthocyanin (node and internode ofthe stem) 2. Node anthocyanin 3. No anthocyanin Pod dehiscence 1. Dehiscent 2. Non - dehiscent Seed testa colour & lustre 1. Black speckled or dark brown, dull 2. Light brown or tan, shinny Disease reaction 1. Apparent immunity to powdery mildew 2. Susceptibility to powdery mildew Tuber form 1. Simple 2. Compound 3. Forked * Observed in cpr 69030 x Ace 390 population only

Table 6.2 Quantitative traits observed in the inheritance studies with cultivated Bali x wild and with var. macrospenna x wild hybrid populations of V. vexillata.

Traits Measurement Size ofterminal leaflet on 6th trifoliolate leaf mm; length & width Stem thickness at primary node, at 6th trifoliolate leafstage mm; diameter Flowering Date when first flower opened First mature pod Date when first pod ripened Seed yield per plant g/plant for first flush ofpods Dry above-ground biomass g/plant Fresh weight oftuberous root at final harvest g/plant Dry weight oftuberous root at final harvest g/plant Tuber diameter at widest point mm; per plant Weight of 100 seeds g/100 seeds Number ofseeds per pod 10 pods per plant; random Pod length and width at pod maturity mm; first 5 mature pods per plant Number offlowers per peduncle 5 peduncles per plant; random Number ofpods per fertile peduncle 5 peduncles per plant; random

94 For the qualitative traits (Table 6.1), the goodness of fit of a model assuming the simplest case of single gene control was first tested (Griffiths et al. 2008). The segregation ratios that were observed in the three segregating generations (F2, BCp!> BCP2) were compared wilh expeclalions from the model for single gene control. If the Chi-squared probability was <0.90, digenic control was also tested. The goodness of fit was tested using GENSTAT IV and SPSS 16.0 Graduate Student Version software (SPSS, Inc)

For the quantitative traits (Table 6.2), broad sense and nan-ow sense heritability were calculated by the variance ratios method (Allard 1999). Broad sense heritability is estimated on the basis of all genetic effects, whereas nalTOW sense heritability is estimated solely using an estimate of additive variance (SIeper and Poehlman, 2006). Variance of each component in the variance ratios method was calculated using SPSS 16.0 Graduate Student Version software (SPSS, Inc). 2 Broad sense heritability is estimated from the formula: h b = VGNp, whilst nalTOW sense 2 heritability is estimated from the formula: h n = VANp (Sieper and Poehlman, 2006).

Two F I hybrids between the var. macrosperma accession CPI 69030 and wild accessions, one from Africa and one from Australia (Table 4.2b), were selected for further observation, together with the respective parental genotypes, for the qualitative and quantitative traits measured in

Experiment 1 (Table 6.1,6.2). For each of these two genotype combinations, four individual F 1 plants, together with four plants of each of the parents, were grown in the shade house. The hybrid and parental genotypes were an-anged in rows on the bench top, with each genotype moved two times during the experiment, and individual plants within each genotype moved every two weeks, in order to reduce possible spatial effects. Simple ANOVA analyses were performed using SPSS 16.0 Graduate Student Version software (SPSS, Inc) to assess the extent of variation for the qualitative and quantitative traits among the parental and F 1 genotypes. In the event, it was not possible to obtain a complete set of data observations for the hybrid combination CPI 69030 x CPI 60458, due to photoperiod sensitivity. In the subsequent analyses, where data for CPI 69030 and CPI 60458 were missing, data for these accessions from Experiment 1 were used instead. In this same cross, complete data were obtained from only one of the four F 1 plants, because like CPI 60458, the other F1 plants failed to flower before the experiment had to be terminated.

95 6.3 Results and discussion 6.3.1 Population development As previously discussed in Chapter 4, population development was carried out on three hybrid combinations, namely, Bali cultivated x Bali cultivated accessions, wild x Bali cultivated accessions, and var. macrosperma x wild accessions. Because there were almost no genotypic differences identified either within or between the different provenances of the Bali accessions (see Chapter 3), it was considered that there would be very limited useful information to be gained from further studies of those hybrid populations. Therefore, no further evaluation of those hybrid populations was undertaken. The results of the population development studies with the other hybrid populations are outlined below.

6.3.1.1 Backcross populations involving Bali cultivated and Australian wild accessions Apart from providing the opportunity to establish the extent that hybrid fertility could be restored through backcrossing, the backcross progeny from the Bali cultivated x Australian wild hybrids provided an opportunity to gain some preliminaly information on the inheritance of cultivated vs. wild traits in V vexillata. Two hybrid populations were available for this purpose:

ACC 390 x Jim lA and ACC 390 x Jim 2. For these F I hybrids, a limited number of successful backcrosses to both the cultivated and wild parent were achieved for later study of the inheritance ofcultivated vs. wild traits.

Backcrosses that attempted to use the Bali x wild hybrid plant as the pollen donor were invariably unsuccessful, regardless of the particular combination of genotypes that was involved, regardless of whether the wild parent or the Bali parent was the pollen receptor, and regardless of whether the pollinations were done in the shade house or in the open garden environment. In all instances, the cross-pollinated flowers abscised 1-2 d after pollination. No evidence ofpod development was observed from many (> 50) cross pollinations.

In contrast, most of the pods from the attempted backcrosses that used either the wild or cultivated parent as the pollen donor and the F I hybrid as the pollen receptor, showed some level ofdevelopment, even though most subsequently abscised within ten days after pollination (DAP) (Figure 6.1). The pattern of pod development / abscission was similar in the two different locations where the backcrosses were attempted, the shade house facilities (Figure 6.la-d) and the open garden environment (Figure 6.le), with the highest number of pods abscising around 5 - 6 DAP. Likewise, the pattern of pod development / abscission was similar regardless of whether the wild or cultivated parent was the pollen donor, or whether Jim lA or Jim 2 was the cultivated parent.

96 :! ;I======jl~[]~p~O~ds~s~U~m~'Y~al~ar~te~r~PO~I~lin~~~io~n~- .1 :t I:~:~~------­ 1113~1::;1.------. - -: -: -: ------~! ~" ~, ~,~ ~'W'?"''''''CZ'' ,:: !, [:,:' ,:: z:r"m,'ftj ftj""*;''''''i'Z»', ...... m OJ,.., d,"ftj' I 2 3 4 S 6 7 8 9 10 II 12 13 Da)'s14 15aftcr16 17po Ilinatio18 19 20n(DAP)21 22 23 24 25 26 27 28 29 30 3 I J2 H 34 35

I 2 3 0; 5 6 1 8 9 10 II 12 13Days 14 ISaftcrl6 17pollinationl8 19 20 (DAP)21 12 23 24 25 26 27 28 29 30 3] 3Z 33 34 31

, tI Pads survival after pollin;~-iOnJ-

I 2 3 4 S 6 7 8 9 10 II 12 13 14 IS 16 17 18 19 20 21 n 23 24 2S 26 27 2R 29 30 31 32 33 34 3S Dnys nftcr pollin.tion (DAP)

10 - ID Pods survival aflcr po LUmuion j.

--- AI fJ ~ ~ r::lIRlI ~RlI~~ I 2 3 4 S 6 7 S 9 10 II 12 IJ Days14 ISafter16 17pollinationIS 19 20 (DAP)21 22 23 24 25 26 27 28 29 30 31 J2 33 34 35

90 I D Pods survival afterpollination 80 I I- 70

60 - 50 I 40 30 20 ~ 10 'IJ- ~ ~ ~"'rJl ll!I

I 2 3 4 5 6 7 8 9 10 II 12 Il 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 )0 31 J2 33 34 3S Da~'s after pollination (DAP)

Figure 6.1 Pod survival time after pollination recorded when cultivated Bali x wild Australian hybrids were backcrossed to the parents, using the respective parent as the pollen donor: a. ACC 390 to ACC 390 x Jim lA in the shade house; b. Jim lA to ACC 390 x Jim lA in the shade house; c. ACC 390 to ACC 390 x Jim 2 in the shade house; d. Jim 2 to ACC 390 x Jim lA in the shade house; e. Jim 2 to ACe 390 x Jim lA in the open garden environment.

97 Interestingly, very few flowers abscised within 1-2 DAP where the F 1 hybrid was used as the female parent (Figure 6.1). This suggested that in most instances, the cross-pollination process was successful and that some pod development was stimulated as a result offertilisation of one or more ovules. Presumably, the subsequent abscission ofmost pods occuned as a consequence of the collapse of the developing seed embryos post-fertilisation. This pattem contrasted with the failure of the F 1 pollen to stimulate any pod development when transfened to the parent flowers which invariably abscised within 1-2 DAP. The latter response was not surprising in view ofthe low viability ofthe F 1 pollen as described in Chapter 5.

The backcross pods that survived beyond 12 DAP matured between 23 DAP and 31 DAP for the backcrosses in the shade house (Figure 6.1a-d), and between 23 DAP and 35 DAP for the backcrosses in the open garden environment (Figure 6.1 e). In most instances, these pods contained only one or two viable backcross seeds, although in a small number of instances, up to 4 viable seeds were obtained. A frequency distribution of the numbers of seeds per pod, summed across the five populations shown in Figure 6.1, is shown in Figure 6.2. The fact that most of the pods were single-seeded (Figure 6.2) implies that one viable seed was sufficient to prevent abscission, at least in these instances.

The small number of viable backcross seeds indicated that there was a high level of hybrid breakdown in those pods that survived to maturity, with the overwhelming majority ofembryos failing to survive post-fertilisation. No attempt was made to dissect the small pods of the F 1 hybrid to count the number of ovules per pod. The mid-parent mean for seeds per pod based on Experiment 1 was 17 seeds (see chapter 3). Using this mean as an estimate of the potential backcross embryos would imply that for those backcross pods that survived to maturity (Figure 6.2), 10% of embryos survived. When the population of backcross pods that did not survive to maturity is considered (Figure 6.1), only 1% ofpossible embryos survived.

2 4 Number of seeds per pod

Figure 6.2 Frequency distribution of the numbers of seeds per backcross pod observed in cultivated Bali x Australian wild hybrids.

98 6.3.1.2 Hybrid population bet-ween var. macrosperma and Austi'alian wild accessions As discussed in Chapter 3, there was considerable similarity observed in Experiment 1 between var. macrosperma CPI 69030 and the Bali cultivated accessions for a range of traits typically associated with domestication, e.g. larger seed size, non-dehiscence of pods, thick stems, broad leaflet shape and large leaflet size. Therefore, the hybrid combination of var. macrosperma cpr 69030 x wild Australian accession ACC 390 provided a useful model for further exploring the inheritance of wild vs. cultivated traits, complementing the evaluation of the wild Australian x Bali cultivated backcross populations.

Backcrosses and F2 generation development were carried out between May 2, 2008 and September 14,2008. There was almost no difficulty in obtaining backcross seeds (about four to eight seeds per backcross pod) and self-F. seeds for F2 generation (about seven to 11 seeds per pod), with the only exception being a limited number of cpr 69030 flowers readily available to backcross to the Fl. Because of the lack of suitable CPI 69030 flowers when the F 1 hybrid was flowering, fewer BCP1 seed were obtained than planned. Thus, fewer than planned BCP. seeds were available for sowing when the time came to sow the main experiment exploring the inheritance I heritability of the cultivated vs. wild traits. Additional backcrosses were made to obtain more BCP1 seed when a later flush offlowers became available on the CPI 69030 parent. These additional seeds were of necessity sown later than the main population. As discussed in

Section 6.2.3, the additional later sown BCP1 plants were used to provide additional infonnation on qualitative tTaits only.

6.3.2 Phenotypic attributes in the Bali cultivated x Australian wild backcross populations

Because the FI plants of the Bali cultivated x Australian wild hybrid populations were unable to self to produce F2 seed, the chi-square goodness of fit test was performed only using the backcross data, to compare the segregation ratios of selected qualitative traits, which were observed in the backcross populations, with the expected model ratios. The goodness offit was considered to be statistically significant (i.e. P < 0.05) where the probability of a larger chi­ square value than that observed was> 0.95. In the first instance, the simplest model of single gene control was tested for each trait, as is illustrated in Table 6.3 for the inheritance of broad vs. nalTOW leaflets in the ACC 390 x Jim IA backcross population.

Even though there were no F2 generation observations available, the results of the chi-square test for both of the ACC 390 x Jim IA and ACC 390 x Jim 2 hybrid combinations (Table 6.4), were consistent with the model that leaflet shape was controlled by a single dominant gene, as

99 reported by James and Lawn (1991). The wild type lanceolate shape in Ace 390 was apparently dominant to the ovate shape in the Bali cultivated accessions. Meanwhile, in terms of leaflet size, which was measured as terminal leaflet width, again the wild leaflet size (narrow size) was apparently controlled by single dominant gene, which was apparently completely dominant to the cultivated leaflet size (broad size) (Table 6.4). This result was different to the study of James and Lawn (1991) which found that the broad leaflet trait of African wild accessions was completely dominant to the nan-ow leaflet type ofAustronesian wild accessions.

Table 6.3 The model used to test the hypothesis of segregation ratios based only on the Bali cultivated x Australian wild backcross populations. The hypothesis was that lanceolate leaflet shape was conditioned by a single dominant gene in the cross between ACC 390 (P t ) and Jim lA (P2)'

Generation Traits Observed (0) Expected (E) Deviation (D) D2/E Backcross to PI (ACC 390) Lanceolate 7 7 0 0 Ovate 0 0 0 0 Lanceolate 6 5 I 0.2 to P2 (Jim lA) Ovate 4 5 -1 0.2 Sum: 004 Probability (P) ofa larger Chi-Square value> 0.99

Table 6.4 Number of individuals within the different phenotypic classes in each generation and the chi-square values for leaflet shape and leaflet width traits observed in two Bali cultivated x Australian wild hybrid popUlations (a) ACC 390 (PI) x Jim lA (P2) and (b) ACC 390 (p.) x Jim 2 (P2).

Par"ental Number of individuals in each generation ·l Traits morphotypesl PI P2 FI Bept BCP2 a. ACC 390 x Jim lA Lanceolate 6 0 6 7 6 Leaflet shape 0040 Ovate 0 6 0 0 4 Narrow 6 0 6 7 5 Leaflet width 0.00 Broad 0 6 0 0 5 b. ACC 390 x Jim 2 Lanceolate 6 0 6 8 Leaflet shape 1.476 Ovate 0 6 0 0 Narrow 6 0 6 8 Leaflet width 2.061 Broad 0 6 0 0

100 As was observed in Experiment I, the Bali cultivated accessions were strongly photoperiod­ sensitive, and they again failed to flower in this evaluation, which was sown as days were lengthening in late spring. In contrast, the Australian wild accession produced flowers within 38 to 40 DAS (Table 6.5). Likewise, all ofthe FI plants from the Bali cultivated x Australian wild hybrid combinations flowered - within 47 to 55 DAS in ACC 390 x Jim lA and 49 to 56 DAS in ACC 390 x Jim 2 (Table 6.5). Again as was observed in Experiment 2, there were no pods set on the F I plants, even when 'tripping' was undertaken to ensure pollen was released onto the floral stigma.

Six ofthe seven BCP 1 plants from the ACC 390 x Jim lA population, and six ofthe eight BCP 1 plants from the ACC 390 x Jim 2 population flowered during this experiment. In contrast, only three of the ten BCP2 plants of the ACC 390 x Jim IA and nine of the 41 BCPz plants of the ACC 390 x Jim 2 flowered. When considered together with the response of the F] generation, the data would seem to suggest that additive gene action for time to flowering. Time to flowering of the F I plants was within the parental range, although it appeared that the performance ofthe progeny was closer to that of the wild parent. When the F1 was backcrossed to the earlier (wild) parent, more of the progeny flowered, whereas when the F] was backcrossed to the later (cultivated) parent, only a small number of progeny flowered. The fact that some BCP2 plants flowered indicated that it may be possible to undertake a backcross program using the cultivated Bali accession as the recurrent parent, but with selection for earliness to flower, to transfer the earliness trait from the wild to the cultivated germplasm. As such, it is likely that the wild material could potentially provide a genetic source of earliness which would obviate the strong photoperiod sensitivity ofthe Bali cultivated accessions.

Ofthe BCP t progeny from the wild x Bali populations, only two plants from each combination set pods successfully. Three ofthese plants set pods only when 'tripping' was conducted, whilst one ofthem set pods freely. All ofthe BCPzplants from both populations failed to set pods even when 'tripping' was conducted. This response suggested that hybrid fertility could be restored through backcrossing to the wild parent.

It was not clear whether the failure for hybrid fertility to be restored through backcrossing to the Bali cultivated parent was absolute, or whether it may have been due in part to environmental effects such as temperature or photoperiod. For example, the failure may have been caused by high temperatures, which were constantly above 35°C during flower development period (about week 5 to week 9 - Figure 6.2) reducing the pollen viability in the BCP2 plants. Exposure to high temperatures during the flower development period (pre-anthesis) can reduce pollen viability in groundnut (Prasad et al. 1999) and common bean (Porch and Jahn 2001).

101 Alternatively, pod set in the BCP2 plants may have been suppressed by the lengthening days, given the generally greater sensitivity ofthe cultivated Bali genotypes to longer photoperiods.

45.0 .,..------

40.0 ~-=;:::==:;::==ii==::a:::::::;:==:;;:=::a::=:;== I ..--. •• .---••- ...... I----I.----l•.--~.------35.0 +-=------G~ 30.0 +---- ~ ~ 25.0 i------_---~-r-----~L------~----=~==----:=:...;=:::::;..~==::;a..~:::;....:- ~ 2o.0+1-"'---;-"~ .. ~~ ~ 15.0 +------""------

Eo< 10.0 +-il------C:=w::i.:1:-:::-;;;:ii:'"::-::l-.-Weekly mean daily minima 5.0 +I------~I Weekly mean daily maxima - 0.0 +---,----.-~--~-_._____-___.__-~-_____,--_.___-~-----,----~--.______, 2 3 4 5 6 7 8 9 10 II 12 13 14 Weeks after sowing

Figure 6.2 Weekly means for daily minimum and maximum temperatures in the shade house during evaluation ofACe 390 x Jim lA and ACC 390 x Jim 2 backcross populations.

Given that only 9 of the 41 BCPz plants flowered before this experiment was necessarily telminated, it is possible that some self-fertile plants may have been identified in the remaining late flowering plants. Regardless, given that it was possible to produce viable BCPz plants, it is likely that further backcrossing to the cultivated parent would likewise be successful, and that over subsequent backcrossing generations, fertility would have been restored.

In tenns of numbers of flowers per peduncle, the F I plants exhibited three to four flowers per peduncle, which was intennediate between the wild Australian genotype (mean 1.8 flowers per peduncle, Table 6.5) and the Bali cultivated parents (mean 8.3 flowers per peduncle, Table 3.7). The numbers offlowers per peduncle in the BCP!, for both hybrid combinations, were similar to the FI, whilst the numbers of flowers in the BCPz was slightly closer to the Bali cultivated parent (Table 6.5). This might suggest that after the hybridisation between cultivated Bali accessions and wild accessions, for instance to transfer the photoperiod insensitivity, number of flowers per peduncle of the cultivated Bali parent could be recovered by backcrossing to the cultivated parent.

102 Table 6.5 Putative quantitative traits observed in parents and their progenies for the ACC 390 (Pt) x Jim lA (P2) and ACC 390 (PI) x Jim 2 (P2) backcross populations. n. a. = dat(l/lOt (IVai/able, I/.m. = data /lot measured.

PI Pz F, BCPt BCPz Traits Mean Range Mean Range Mean Range Mean Range Mean Range a. ACC 390 x Jim lA i. Morphological traits Stem thickness (mm) 1.8 I.7 - 1.9 n.m 2.7 2.4 - 3.3 2.4 2.4 -2.6 3.5 2.6 - 5.0 Terminal leaflet length (mm) 166.1 143.4 -175.4 n.m 137.4 JlO.7-160.8 147.1 115.6 - 224.4 141.7 124.5 - 158.4 Terminal leaflet width (mm) 10.1 8.5 - 11.2 n.m 21.8 16.4 - 28.8 21.6 18.9 - 25.1 61.9 30.7 - 80.6 Leaflet length/width ratio 16.6 15.2 - 17.4 n.m 6.4 5.6 - 7.0 6.8 5.4 - 10.4 2.9 1.7 - 5.2 Flowers per peduncle 1.8 1.6 - 1.8 n. a 3.5 3.0 - 4.2 3.4 2.4 - 3.8 5.8 3.0 - 7.3 Pods per fertile peduncle 1.7 1.6 - 1.8 n. a n. a 1.5 1.5 n.a Pod length (mm) 110.5 107.4-114.2 n. a n.a 56.6 56.6 n.n Pod width (mm) 4.2 4.2 n.a n.a 4.8 4.8 n. a Seeds per pod 19.7 19.5 - 20.1 n. a n.a 1.3 1.0 - 1.6 n.a 100 seeds weight (g) 1.3 1.2 - 1.3 n. a n. a 1.8 1.5 - 2.1 n.a Tuber diameter (mm) 13.6 11.4-17.9 8.1 5.7 - 11.2 18.7 14.9 - 22.5 16.4 12.0 - 24.2 13.1 7.3 - 20.4 ii. Agronomic traits Dry above-ground biomass (g) 25.7 14.0 - 34.0 54.7 46.0 - 64.0 41.2 12.0 - 82.0 46.6 14.0 - 80.0 45.6 14.0 - 86.0 Total dry matter (TDM) (g) 42.6 35.9 - 50.1 n.a n. a 69.1 52.1 - 86.0 n.a Total seed yield per plant (g) 9.9 9.6 - 10.3 n. a n. a 0.1 0.1 n.a Tuber fresh weight (g) 46.0 32.0 - 62.0 22.0 18.0 - 28.0 73.6 48.0 - 110.0 62.3 28.0 - 84.0 41.8 18.0 - 98.0 Tuber dry weight (g) 7.0 4.0 - 12.0 1.0 0.0 - 2.0 12.8 6.0 - 22.0 10.3 2.0 - 16.0 4.4 0.0 - 18.0 Seed harvest index (SRI) 0.3 0.2 - 0.3 n. a n.a 0.0 0.0 n.a Tuber harvest index (TRI) 1.1 0.7 - 1.7 n. a n. a 1.2 1.0 - 1.3 n.a iii. Phenological traits Flowering (d after sowing) 40.0 38 - 43 n. a 52.4 47 - 55 53.3 45.0 - 82.0 61.3 57 - 68 First mature pod (d after sowing) 56.2 54 - 60 n. a n.a 80.5 80 -81 n.a Seed development period (d) 16.2 16 - 17 n. a n. a 17.5 17 - 18 n. a Table 6.5 eon/.

Traits PI P2 F1 BCP\ BCP2 Mean Range Mean Range Mean Range Mean Range Mean Range b. ACC 390 x Jim 2 i. Morphological traits Stem thickness (mm) n.m 2.7 2.4 - 3.1 2.3 1.9 - 2.5 3.8 3.0 - 5.2 Terminal leaflet length (mm) n.m 130.7 118.4 -145.2 158.5 141.5-181.4 167.1 132.1 - 194.4 Tenninalleaflet width (mm) n.m 23.6 21.0 - 29.4 15.0 10.2 - 18.6 41.2 30.0 -75.8 Leaflet length/width ratio n.m 5.6 4.6 - 6.9 11.1 7.7 - 14.0 4.7 1.7 - 6.5 Flowers per peduncle n.a 3.6 3.2 - 4.0 2.8 2.0 - 4.0 4.4 3.0 - 5.7 Pods per fertile peduncle n.a n. a 1.5 1.4-1.6 n.a Pod length (mm) n.a n. a 68.6 56.9 - 80.3 n.a Pod width (mm) n.a n. a 3.7 3.6 - 3.7 n.a Seeds per pod n.a n. a 6.1 3.9 - 8.2 n. a 100 seeds weight (g) n.a n. a 1.1 0.9 - 1.3 n. a Tuber diameter (mm) 5.0 1.7 - 8.2 27.5 24.1 - 37.2 25.5 18.2 -35.5 11.1 4.1 - 20.0 ii. Agronomic traits Dry above-ground biomass (g) 52.3 46.0 - 60.0 42.5 28.0 - 50.0 30.8 14.0 -52.0 48.2 6.0 - 100.0 Total dry matter (TDM) (g) n.a n. a 42.6 40.9 - 44.3 n.a Total seed yield per plant (g) n. a n. a 0.6 0.3 - 0.9 n. a Tuber fresh weight (g) 12.0 8.0 - 16.0 65.5 56.0 -72.0 89.5 52.0 - 150.0 31.7 12.0 - 64.0 Tuber dry weight (g) 0.0 0.0 14.0 10.0 - 16.0 19.5 10.0 - 30.0 3.2 0.0 - 10.0 Seed harvest index (SH1) n. a n. a 0.0 0.00 n. a Tuber harvest index (THI) n. a n. a 2.4 1.4-3.4 n. a iii. Phenological traits Flowering (d after sowing) n. a 51.8 49 - 56 46.2 39 - 52 54.8 39 - 61 First mature pod (d after sowing) n. a n.a 68.5 65 -72 n.a Seed development period (d) n. a n. a 16.0 16 n. a

104 In this evaluation, the Bali cultivated parents had small values for tuber attributes, compared to the wild parent and the F(, F2 and backcross generations (Table 6.5). This result was surprising, given that the Bali accessions are grown for their tubers. However, it is likely that the small tuber growth of these accessions was an artefact of the environmental conditions under which the plants were grown in this evaluation. Days were lengthening through most of the growing period and flowering in the Bali accessions was completely inhibited. In other quantitative short day plants, tuber formation is promoted under short photoperiods, as reported by Okubo et al. (1992) in winged bean (Psophocarpus tetragonolobus). In V. vexillata, Karuniawan and Lawn (2007) reported that artificial day length extension promoted vegetative growth, and reduced partitioning ofdry matter into tubers.

6.3.3 Evaluation of selected attributes in var. macrospel'ma x wild African hybrids Generally, all the accessions and their hybrids that were evaluated in these studies exhibited ovate leaflet shape and broad leaflet size, but a mixed reaction to powdery mildew (Table 6.6). The fact that even the parents showed a mixed reaction to powdery mildew suggested that the observed differences were probably due to chance escape ofinfection by some plants. Based on the observations presented in Table 6.6, it appeared that dehiscent pod was dominant over non dehiscent pod, because both of the hybrid combinations between var. macrosperma (which exhibited non dehiscent pods) and the two wild African accessions (which exhibited dehiscent pods) had dehiscent pod attributes. A similar result was also observed for the seed testa pattern attribute, where black speckled was apparently dominant over uniform seeds (Table 6.6).

Table 6.6 Six qualitative traits observed in var. macrospel'ma x wild African hybrids and their parents.

P2 FI PI FI P2 Traits (CPI (CPI 69030 x CPI (CPI (CPI 69030 x CPI (CPI 29141) 29141) 69030) 60458) 60458) Leaflet shape Ovate Ovate Ovate Ovate Ovate Leaflet width Broad Broad Broad Broad Broad Leaflet hair Long Long Long Short Short length Pod Non Dehiscent Dehiscent Dehiscent Dehiscent dehiscence dehiscent Seed testa Speckled Speckled Unifonn Speckled Speckled pattern Disease Mixed Mixed Mixed Mixed reaction Mixed reaction reaction reaction reaction reaction

105 Some quantitative traits were measured on the cpr 69030 x cpr 29141 and cpr 69030 x cpr 60458 hybrids and their parents. Simple ANOVA and Least Significant Difference (LSD) tests were performed to test the level ofdifferences among the cpr 69030, cpr 29141, and F1 means and to estimate whether the F1 performance for a specific trait was closer to the male parent (Cpr 69030) or the female parent (Cpr 29141) (Table 6.7).

Table 6.7 Putative quantitative traits observed in parental morphotypes and their hybrid for the CPI 69030 x CPI 29141 hybrid population. Means within a row followed by the same letter are not significantly different (P > 0.05).

PI Pz Traits FI (CPt 69030) (CPt 29141) a. Morphological traits Stem thickness (mm) 4.8 a 2.6 b 2.4 b Tenninalleaflet length (mm) 130.9 b 103.0 c 142.8 a Tenninalleaflet width (mm) 53.8 b 64.8 a 59.4 b Leaflet length/width ratio 2.4 a 2.2 b 1.7 c Flowers per peduncle 3.5 a 3.7 a 3.7 a Pods per fertile peduncle 2.7 a 2.8 a n.m Percentage ofsetting pod (%) 75 a 77a n.m Pod length (mm) 137.3 a 102,4 b 94.2 c Pod width (mm) 5.9 a 5.2 b 4.1 c Seeds per pod 18.5 a 11.5 c 16.9 b 100 seeds weight (g) 5.3 a 3.4 b 1.7 c Tuber diameter (mm) 22.9 a 18.7 b 11.5 c b. Agronomic traits Dry above-ground biomass (g) 7.0 b 16.0b 58.0 a Total dry matter (TDM) (g) 37.1 b 66.4 a 39.5 b Total seed yield per plant (g) 16.1 a 13.5 b 5.9 c Tuber fresh weight (g) 64.5 a 42.0b 29.0b Tuber dry weight (g) 15.5 a 1O.0b 2.5 c Seed harvest index (SHI) 0.4 a 0.3 b 0.1 c Tuber harvest index (THI) 1.6 a l.lb 0.5 c c. Phenological traits Flowering (days after sowing) 44.0 a 51.7b 73.5 c First mature pod (days after sowing) 66.0 a 69.7 a 92.0b Seed development period (days) 22.0 b 18.0 a 18.5 a

A complete set of data for the 69030 x cpr 60458 cross was not obtained (Table 6.7) because the wild African parent, cpr 60458, and three of the four putative F1 hybrid plants, were

106 strongly photoperiod sensitive and did not flower within the experimental period. For comparative purposes, some data for reproductive stage attributes of CPI 60458 were substituted with results from harvest 1 Experiment 1. While useful for comparative purposes, it was not appropriate to analyse these data statistically, due to possible environment (E) effects and genotype x environment (G x E) interaction effects between the two studies.

For most of the quantitative traits, the F I plant phenotype was intermediate between the parental lines, CPI 69030 and cpr 29141, for instance, terminal leaflet length/width ratio, pod length, pod width, 100 seeds weight and tuber diameter (Table 6.7). Meanwhile in some traits, e.g. tenninal leaflet width, and TDM, the F I plants had higher values than the higher parent value, whilst for terminal leaflet length and seeds per pod, the F I plants had lower values than the lower parent value (Table 6.7).

Number ofpods per fertile peduncle and percentage of setting pods were only calculated in the cpr 69030 and the FI plants, because the flowers ofcpr 29141 were 'tripped' by hand to obtain pods and seeds, due to general failure to set pod in the shade house as described in Experiment 2 (Chapter 4). It suggested that the degree of self-pollination was presumably heritable qualitatively. There were no significant differences between CPI 69030 and the FI in those two traits. This was ofinterest because like cpr 29141, the cultivated Bali accessions also exhibited lower levels ofself-pollination as observed in Experiment 2 (Chapter 4).

There was some degree of dominance observed in stem thickness, first mature pod and seed development period, as indicated by the F I performance being relatively closer to one parent compared to the other parent (Table 6.7). Even though there were significant differences between those three generations in flowering time, the F I performance was relatively closer to cpr 69030 in tenns of the first mature pod trait, because the F I plants had a shorter seed development period than cpr 69030, which was similar to cpr 29141 (Table 6.7).

For many traits, the performance of the F I plant of the CPI 69030 x CPI 60458 cross were also intermediate between the two parental lines (Table 6.8), as observed in the FI plants ofthe cpr 69030 x CPT 29141 cross (Table 6.7). In some traits, such as pod length, pod width, and 100 seed weight, the F I performance was consistently intermediate between the two parental lines in both these hybrids combination, whilst in number of seeds per pod, the F I performance was lower than the lowest parent lines. From those data, it would seem to suggest that additive gene action for that pod length, pod width and 100 seed weight. Meanwhile, even though it was suggested that additive gene action was observed for pod length, it appeared that the

107 perfonuance of the progeny was closer to that of the parent that exhibited the shorter pods. Presumably, the increase in seed size was not accompanied by a significant increase in pod length, as a consequence, lower numbers of seeds per pod were observed in both hybrid combinations (Table 6.7, 6.8). It suggested that there was a small degree of dominance in that trait, as reported by Leleji (1975) in cowpea that short pod was partially dominant over long pod.

Table 6.8 Putative quantitative traits observed in parental morphotypes and their hybrid for the CPI 69030 x CPI 60458 hybrid popUlation. The values were the mean values for each trait in each generation.

PI P1 Traits FI (CPI69030) (CPI60458) a. Morphological traits Stem thickness (mm) 7.0 4.2 2.3 Terminal leaflet length (mm) 144.8 116.0 77.0 Terminal leaflet width (mm) 88.5 69.3 43.7 Leaflet length/width ratio 1.6 1.7 1.8 Flowers per peduncle 4.0 3.4 2.7 Pods per fertile peduncle 2.9 2.6 1.9 1 Pod length (mm) 147.61 105.0 89.5 1 Pod width (mm) 6.4 1 5.0 4.1 1 Seeds per pod 19.i 9.4 l1.i 100 seeds weight (g) 4.89 1 3.6 1.0i Tuber diameter (nun) 8.9 5.4 3.5 b. Agronomic traits Dry above-ground biomass (g) 52.5 44.0 29.7 Tuber fresh weight (g) 28 38 11 Tuber dry weight (g) 3.5 6 0 c. Phenological traits Flowering (days after sowing) 84.7 58.0b 96.0 Seed development period (days) 16.3 3 16.0 15.0' • Values were obtained from harvest 1Experiment I data; b Only 1F1 plant flowered

As was observed in the Bali cultivated x Australian wild backcross populations (Section 6.3.2), tuber vs. shoot and flower formation in the two populations ofvar. macrosperma x African wild hybrids appeared to be affected by photoperiod. The cpr 69030 x cpr 60458 evaluation experienced longer days (>14 hours) than did the cpr 69030 x cpr 29141 evaluation, which was sown earlier. The values for tuber attributes of cpr 69030 in the cpr 69030 x cpr 60458 evaluation (Table 6.8) were lower than the values in cpr 69030 x cpr 29141 evaluation (Table

108 6.7). In contrast, dry above-ground biomass of CPI 69030 was higher in the CPI 69030 x CPI 60458 evaluation, compared to the CPI 69030 x CPI 29141 evaluation. Meanwhile, the lengthening days during summer in the evaluation ofCPI 69030 x CPI 60458 delayed flowering in both parental lines and the three of the four the F1 plants. Interestingly, one of the four F 1 plants, which flowered during evaluation, flowered earlier than both parental lines (Table 6.8). This might suggest that heterosis in time to flowering occurred in this hybrid combination.

In order to obtain complete data for all four F I plants in the hybrid population of CPI 69030 x

CPI 60458, the three F 1 plants that had not flowered by the end of the experiment were pmned back as outlined in Section 3.2.4. However, approximately 3 weeks after they were pruned back, those F I plants died, perhaps because of the limited number of internodes on the 20 em stern above the medium where, on other genotypes, the new shoots usually develop after being pmned back.

6.3.4 Inheritance of selected traits in val'. macrosperma x wild Australian hybrids The val'. macrosperma x Australian wild accession ACC 390 hybrid population was chosen for the inheritance studies, rather than the two other hybrid combinations of val'. macrosperma X African accessions, in part because ACC 390 was used in the hybrid populations with the Bali cultivates accessions. In addition, expression of many of the wild traits in ACC 390 was more extreme that in some of the other wild accessions, providing greater differentiation from the cultivated attributes exhibited by val'. macrosperma. This enhanced the ability to discriminate between phenotypes and made observation of segregation easier, as suggested by Mendel (1866).

6.3.4.1 Weather conditions The seasonal profiles of mean weekly maximum and minimum temperatures during the plant growth period for this study are shown in Figure 6.3. The main group of plants, sown on September 17, 2008, had all produced their first flush ofmature pods by December 13,2008 (13 weeks after sowing). The subset of additional BCP 1 plants, sown on October 25, 2008, did not mature their first flush of pods until January 11, 2009 (week 17). Thus the main population of plants grew during the period spring to early summer (weeks 1-13, Figure 6.3) while the smaller subset grew during late spring to midsummer (weeks 6-17, Figure 6.3). Weekly mean minimum temperatures during weeks 1-13 ranged 15.8 - 24.1°C with occasional daily minima as low as 12°C and as high as 26°C. Weekly mean maximum temperatures ranged 35.6 - 40.7°C, with occasional daily maxima as low as 34°C and as high as 44°C. During weeks 6-17, weekly mean minimum temperatures ranged 21.0 - 24.1°C with occasional daily minima as low as 16°C and

109 as high as 26°C. Weekly mean maximum temperatures for weeks 6-17 ranged 36.7 - 40.7°e, with occasional daily maxima as low as 35°e and as high as Moe.

45.0 -,------

40.0 -;------~ 35.0 i· ····· ·· E30'0~ . .. ~ ------=~=_:_=~:::;;;Il~~;;;;;;;;;;~~;:;;:::;:::;;::::;;:- .. 25.0· .. .-.--* '" '" * • '" ... i 20.0 ~...,~"'---:::::::....~::::------~ 15.0 I 10.0 l------I...... Da~ly mini.ma 1­ 5.0 +J------1_Dally maxlma-

0.0 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 Weeks after sO\ung

Figure 6.3 Weekly means for daily minimum and maximum temperatures during Experiment 4.

The main popUlation was grown between week 1 and week 13. The additional population of BCP j was grown between week 6 and week 17.

Daily photoperiod increased from 12 h 48 min when the main group ofplants was sown to 14 h 6 min at week 13 when these plants were harvested for biomass measurement (Figure 6.4). For the later sown subset, the daily photoperiod had already lengthened to 13 h 30 min when these plants were sown. These plants completed their last five weeks of growth when daily photoperiods were longest (> 14 h, Figure 6.4).

14.50.------

'",' 14.00 ------:--=_...... ,~~:!::==!::~~~~~.- ~ 5 ~--- f 13.50 +------.....-.=------'1: ~ ~ 13.00 +-~------:~.-="------

f 12.50 ·1------

12.00 +--,------;----,-----,-----,-----.----,----,----,------,------,------.------,----,-----.---,---~ 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ]6 17 Weeks after sowing (weeks)

Figure 6.4 Weekly means for daily photoperiod (including civil twilight) during Experiment 4. The main population was grown between week 1 and week 13. The additional population of BCP1 was grown between week 6 and week 17.

110 In general, therefore, the later sown subset ofBCP j plants experienced longer photoperiods and wanner conditions for most of their growth period than the early sown plants. Because of the large effects ofphotoperiod on phenology and other attributes of growth observed in the earlier studies, especially in Experiment 1, it was considered likely that there would be large environment (E) and genotype x environment (G x E) interaction effects on the expression of quantitative traits in the two groups ofplants.

This concem was bome out by a comparison of the average time to flowering of the BCP 1 plants in the main population with that in the later sown subset. The earlier sown BCP t plants flowered an average 52 days after sowing (DAS), when daily photoperiod was 13 hours 36 minutes (week 7). The later sown plants flowered an average 57 DAS, when photoperiod was 14 h 6 min (week 14). Delays in flowering time when short day plants are exposed to longer photoperiods are a common OCCUlTence (Caber et al. 2001, Upadhyay et al. 1994). In tum, photoperiod-induced effects on phenology can have large effects on attributes of plant growth and seed yield (e.g. Lawn 1979b, Hadley et al. 1984).

Because of the risk of E and G x E effects on the expression of quantitative traits in the two groups of plants, the measurements of quantitative traits from the later sown subset of BCP t plants were excluded from the subsequent analyses of inheritance of these traits. The data for putative qualitative traits from both populations were used in the analyses ofinheritance, on the basis that by their nature, environmental effects on expression ofthese traits are negligible.

6.3.4.2 Qualitative traits The standard Chi-square goodness of fit test was used to compare the segregation ratios that were observed in all the putative qualitative traits with the expected model ratios (Table 6.9). In the first instance, the simplest model of single gene control was tested for each trait. Where the apparent goodness of fit was not statistically significant (P < 0.05), more complex digenic models of control were evaluated. Coincidentally, most of the qualitative traits that were observed in this study were found to be controlled by single dominant genes (Table 6.10). The only trait where it was considered necessary to evaluate a more complex model was seed testa colour.

For both the leaflet shape and leaflet size traits, the segregation ratios were consistent with the hypothesis of single completely dominant gene control, with "l of 1.993 and 3.250, respectively (Tables 6.10). In tenns ofleaflet shape, the wild type lanceolate shape was apparently dominant to the ovate shape of var. macrosperma, which is usually associated with domestication (Table

111 6.10). This fmding was consistent with the study using African x Australian accessions of V. vexillata by James and Lawn (1991). Likewise, the wild type narrow leaflet size trait was completely dominant to the broad leaflet trait of var. macrosperma. This result was the same with the proposed gene control in wild Austronesian x cultivated Bali hybrid combinations in

Section 6.3.2, but again it was different to the study by James and Lawn (1991). In the F2 generation, independent segregation of the leaflet shape-leaflet width traits was observed, with both narrow and broad leaflets observed within each of the lanceolate and ovate leaflet shape classes (Plate 6.1).

Table 6.9 The model used to test the hypothesis of segregation ratios. The hypothesis was that lanceolate leaflet shape was conditioned by a single dominant gene.

Generation Traits Observed (0) Expected (E) Deviation (D) D2/E

F2 Lanceolate 77 72.75 4.25 0.248

F2 Ovate 20 24.25 4.25 0.745 Backcross

tOPl Lanceolate 15 12.5 2.5 0.5

tOPt Ovate 10 12.5 2.5 0.5

to P 2 Lanceolate 24 24 0 0

to P2 Ovate 0 0 0 0 Sum: 1.993 Probability (P) ofa larger Chi-Square value> 0.99

Because no specific gene nomenclature has been developed for V. vexillata, James and Lawn (1991) proposed that the symbols for gene nomenclature used by Fery (1980) for cowpea (V. unguiculata), should be adopted for V. vexillata. Consistent with that suggestion, the nomenclature used by Fery (1980), and extended by James and Lawn (1991) where novel nomenclature was needed, is used in the following discussion. James and Lawn (1991) adopted the symbols La, la and Blf blf for leaflet shape and size, respectively. Given that the broad leaflet trait in val'. macrosperma was recessive to the narrow leaf shape of the wild Australian gennplasm, whereas in wild African gennplasm it was dominant, there may be more than one allele for broad leaflet size in the V. vexillata gene pooL Broad leaflet size recessive to the narrow leaflet was observed in cowpea and it was proposed to use the symbol Nlf nlffor the traits (Kohle 1970 cited by Fery 1980).

CPI 69030 had very long leaflet pubescence (> 1 mm) that also could be observed on the stems and pods but at a lower density. In contrast, ACC 390 had very short: leaflet pubescence «1 mm) and stem pubescence was terete (See Chapter 3). In tenns of leaflet pubescence, the wild

112 type trait short leaflet hair length (Hs) was apparently completely dominant to long leaflet hair length (hs) (Table 6.10). A similar result was reported by James and Lawn (1991). Plant pubescence in lentil (Kumar et al. 2005) and in black gram (Arshad et al. 2005) was completely dominant to glabrous and controlled by a single gene. It has been suggested that the degree of plant hirsuteness can influence the palatability of plant tissues to grazing animals (James and Lawn 1991). Certainly, it has been shown to affect susceptibility to insect predation (Chiang and Singh 1988; Jackai and Oghiakhe 1989; Fatokun 2002).

Table 6.10 Number of individuals within the different phenotypic classes in each generation and the chi-square values for several putative qualitative traits observed in CPI 69030 (P t) x ACC 390 (P2) hybl'id populations.

Parental Number of individuals in each generation Traits morphotypesa ·l Pt P2 F 1 BCPt BCP2 F2 Lanceolate 0 10 6 15 24 77 Leaflet shape 1.993** Ovate 10 0 0 10 0 20 Narrow 0 10 6 14 24 80 Leaflet width 3.250* Broad 10 0 0 11 0 17 Short 0 10 6 14 24 80 Leaflet hair length 3.250* Long 10 0 0 11 0 17 Anthocyanin 0 10 6 10 24 67 Stem pigmentation 2.818* Non anthocyanin 10 0 0 15 0 30 Dehiscent 0 10 6 7 24 78 Pod dehiscence 2.404* Non dehiscent 10 0 0 11 0 19 Speckled 0 10 6 7 24 67 Seed testa pattern 2.707* Unifonn 10 0 0 11 0 30 Dark brown 0 10 6 7 24 72 Seed testa colourb Light brown-green 10 0 0 11 0 19 0.934** Green 0 0 0 0 0 6 *significantP < 0.05; ** significant at P < 0.01 a apparently dominant parental morphotypes are listed first; b trait apparently under digenic control

There was no anthocyanin observed on CPI 69030 stems, whilst ACC 390 had stem pigmentation but only on the nodal areas between the stem and branches. Stem pigmentation was apparently completely dominant to non-pigmented stem, with a "l value of 2.818 (Table 6.10). Nodal pigmentation, controlled by a single completely dominant gene, has been also reported by Othman et al. (2006) in cowpea. Meanwhile, James and Lawn (1991) rep011ed stem pigmentation in V. vexillata, in terms of full pigmentation vs. nodal pigmentation, was also controlled by single completely dominant gene. James and Lawn (1991) adopted An, an as symbol for that gene.

113 CPI69030 I ACC 390

F2 generation

Plate 6.1 Independent segregation ofthe leaflet shape and leaflet size traits in F, generation plants from the CPI 69030 x ACC 390 hybrid population, compared to the parental phenotypes. Leaflet shape segregated between lanceolate (a, b, c) and ovate (d, e), combined witb some differences in leaflet size. Photographs were taken on the same day at the same magnification.

Pod dehiscence is one of the key distinctive traits that differentiate between domesticated and wild accessions. Pods of CPI 69030 did not dehisce upon maturity, even after they had completely dried, whilst the pods of ACC 390 dehisced as soon as they dried. Segregation of dehiscent vs. non dehiscent pods in the F, generation of the CPI 69030 x ACC 390 hybrids was consistent with the hypothesis ofa single completely dominant gene for the wild type trait, with a X' of 2.404 (Table 6.10). Similar results have been reported in crosses between wild and cultivated cowpeas by Aliboh et at. (1996), and in lentil by Ladizinsky (1979). Aliboh et at. (1996) assigned the labels Dhp, dhp to code for pod dehiscence in cowpea. In addition, pod

114 dehiscence was detected to be controlled by single dominant gene using QTL analysis in V.

angularis (lsemura el af. 2007)

CPI 69030 exhibited a non-speckled light brown-green testa, whilst the wild accession ACC 390 had dark brown testa with black speckles (Plate 6.2). The occurrence of black speckles on the testa was completely dominant to a non-speckled testa, with a x' of2.707 (Table 6.10). This finding was consistent with the observations in African x Australian accessions of V. vexi//ala

(James and Lawn 1991), in black gram (Arshad el al. 2005), and in lentil (Ladizinsky 1979). James and Lawn (1991) proposed Spk, spk as a symbol for the black seed speckle attribute.

Plate 6.2 Independent segregation of seed colours and non-speckled vs. black speckled traits in F, generation plants from the CPI 69030 x ACC 390 hybrid population, compared to the parental phenotypes and the hybrid. Seed testa colour segregated from light brown-green seed (c, d), light brown seed (a, b), and dark brown seed (e), combined with non-speckled (a, c) or black speckled attributes (b, d, e). Black scale bar was 10 mm.

Meanwhile, the F, plants segregated into three classes of seed testa colour (Plate 6.2 F, c-e), which were dark brown, light brown-green and green, in the ratio of 12 : 3 : I, respectively (Table 6.10). All of the F, plants exhibited dark brown seed testa colour. It suggested that seed testa colour in CPI 69030 x ACC 390 was controlled by two pairs of genes in dominance­ epistatic interaction. Seed testa colour in this experiment was observed a week after harvesting the pod. However, seed testa colour was apparently not fully stable, which was indicated by changes to darker colour during storage.

115 6.3.4.3 Linkage between qualitative traits Analysis oflinkage between qualitative traits, which were controlled by single dominant genes, was accomplished by using the Chi-square of goodness of fit. The joint segregation ratios of trait pairs, which were observed in F2 generation, were compared to the expected distribution for independent ass0l1ment (9:3:3:1). Where the deviation was statistically significant (P < 0.05), it suggested that there was an apparent linkage between those two traits.

Linkage was mostly detected in relation to wild traits, which were exhibited by ACC 390, i.e. linkage between stem pigmentation and pod dehiscence, stem pigmentation and seed testa pattern (black speckled), and pod dehiscence and seed testa pattern (black speckled) (Table 6.11). There was an apparent linkage also detected between 1anceo1ate leaflet shape and narrow leaflet width (Table 6.11), which also reported by Grant et at. (2003) in observations of 79 Australian V. vexillata accessions.

Table 6.11 Chi-square values were calculated from six qualitative traits observed in CPI 69030 x ACC 390 hybrid population to detect genetic linkage among those six traits.

Stem Leaflet hair Pod Traits Leaflet shape Leaflet width pigmentation length dehiscence Leaflet width 52.022* Stem pigmentation 4..553 5..543 Leaflet hair length 17.493* 14.853* 5.. 543 Pod dehiscence 4.077 7.156 21.231* 4.737 Seed testa pattern 3.417 4.847 121.667* 5..543 26.107* *, significant at P <0.05

6.3.4.4 Quantitative traits

For many traits, the phenotype ofthe F j plants was intermediate between the parenta11ines, CPI 69030 and ACC 390 (Table 6.12). In some traits, e.g. terminal leaflet length, pods per fertile peduncle, percentage of set pods, TDM, tuber fresh weight and tuber dry weight, the F I plants had higher values than the higher parent value, whilst for pod length, seeds per pod and seed harvest index (SRI), the F I plants had lower values than the lower parent value (Table 6.12).

Large variability was observed for tuber fresh weight, terminal leaflet length, and TOM, whilst the smallest amount ofvariation was observed for Sill, and to a lesser extent, the percentage of set pods (Table 6.11). There was some transgressive segregation observed in the F2 generation

(Table 6.11). For instance, in the time to flowering trait, 12 F2 plants flowered earlier than the earlier parent (ACC 390) and seven plants flowered later than the later parent (CPI 69030) (data

116 not shown). The variation for quantitative traits in the F2 generation was generally continuous, with flowering the main trait showing some evidence ofdiscontinuities in response (Figure 6.5).

Table 6.12 Putative quantitative traits observed in parental morphotypes and their progenies for the CPI 69030 x ACC 390 hybrid population.

F2 Pt P2 Traits F1 cpr 69030 ACC 390 Mean Range a. Morphological traits Stem thickness (mm) 8.4 4.7 2.4 4.8 3.0 - 7.1

Tenninalleaflet length (mm) 134.5 173.7 148.1 158.1 96.3 - 222.0 Tenninalleaflet width (mm) 69.8 20.5 8.2 25.3 7.6 - 67.0 Leaflet length/width ratio 1.9 8.5 18.3 8.1 2.3-21.0 Flowers per peduncle 4.7 4.1 2.6 4.0 3.0 - 5.2 Pods per fertile peduncle 3.4 3.7 2.3 3.3 2.0 - 4.2 Percentage ofsetting pod (%) 73 89 87 82 56 - 95 Pod length (mm) 148.2 102.5 107.4 108.3 71.2 - 147.4 Pod width (mm) 6.6 5.1 4.1 5.1 4.1-6.1 Seeds per pod 20.3 9.2 20.2 13.5 6.1-21.2 100 seeds weight (g) 5.1 3.6 1.4 2.9 1.3 -4.7 Tuber diameter (mm) 16.7 14.3 10.5 15.6 7.8 - 29.5 b. Agronomical traits Total dry matter (TDM) 79.1 103.5 49.1 94.7 43.1 - 155.4 Total seed yield per plant (g) 24.7 23.5 13.5 18.4 10.1 - 33.5 Tuber fresh weight (g) 46.0 55.0 23.6 17.7 12.0 - 252.0 Tuber dry weight (g) 9.8 14.3 4.0 17.7 0.0 - 62.0 Seed harvest index (SHl) 0.3 0.2 0.3 0.2 0.1 - 0.4 Tuber harvest index (THl) 0.6 0.5 0.5 0.7 0.2 - 2.1 c. Phenological traits Flowering (days after sowing) 51.9 49.8 48.3 50.7 43.0 -72.0

First mature pod (days after sowing) 68.5 65.0 63.2 66.6 60.0 - 86.0 Seed development period (days) 16.6 15.7 14.9 15.9 13.0 - 20.0

117 - t - ,"-- -~ ..,--

f~ ~ ~ ~ o ~ 43-4445-46 47-48 49-50 51-52 53-54 55-56 57-58 59-60 61-62 63-6465-6667-68 60-70 71-72 (a) Flowering (days after sowing)

30/ .l!l 25 ,,-~~~~~~~~~~~--.:t:.Si::i:/!I-~~~-~------1 ] 20V ~ 15 V~------DC>Cl')I

~ 10 I § J/ !I~~~~. Z 0-1-

16 V '" 14 12·V ==Ol ....c.. 10 V 0 8.V .. >( • ... 6 V .Cle ::I 4V Z 2~ 0 ~, <11.51 II.5I-IJ.50 13.51.15.50 15.51·17.50 17.51· 19.50 19.51-2J.50 21.51.23.50 23.51-25.50 2550·27.50 (c) Total seed yield per plant (g)

6.01-9.00 9.01-12.00 12.01-15.00 15.01-18.00 18.01-21.00 21.01-24.00 24.01-27.00 27.01-30.00 (d) Tuber diameter (mm)

FigUl-e 6.5 Frequency distl"ibution of 97 F2 plants from the CPI69030 x ACC 390 cross, observed for: (a) flowering; (b) 100 seeds weight; (c) total seed yield per plant and (d) tuber diameter.

118 Based on the heritability classification of Hallauer and Miranda (1988), high broad sense

2 heritability values (h b) were observed in the cpr 69030 x ACC 390 hybrid population for 2 almost all of the traits evaluated, with h b ranging between 0.66 and 0.99 (Table 6.13). For the same traits, low to very high narrow sense heritability values (h2n), ranging between 0.27 and 1.00, were detected (Table 6.13). Some narrow sense heritability estimates actually exceeded 1, which is not possible, but which nonetheless meant that the traits were very highly heritable. Conversely, some zero narrow sense heritability values were also observed, which indicated minimal heritability, perhaps due to a large degree ofdominance genetic variance suggestive of epistatic gene action, or alternatively, linkage disequilibrium (James and Lawn 1991).

2 2 Table 6.13 Broad sense (h b) and narrow sense (h n) heritability values for the putative quantitative traits measured in the CPI 69030 x ACC 390 hybrid population.

Traits S.En a. Morphological traits Stem thickness (mm) 0.89 0.03 0.83 0.15 Terminal leaflet length (mm) 0.85 0.05 0.69 0.]4 Terminal leaflet width (mm) 0.87 0.07 1.001 0.00 Leaflet length/width ratio 0.90 0.05 1.001 0.06 Flowers per peduncle 0.80 0.06 0.37 0.25 Pods per fertile peduncle 0.82 0.07 0.79 0.12 Percentage ofsetting pods (%) 0.78 0.09 1.00 1 0.05

Pod length (mm) 0.97 0.01 0.06 0.45 Pod width (mm) 0.93 0.02 0.72 0.25 Seeds per pod 0.99 0.00 0.002 0.57 100 seed weight (g) 0.88 0.04 0.83 0.22 Tuber diameter (mm) 0.81 0.06 0.78 0.13 b. Agronomic traits

Total dry matter (TDM) 0.66 0.10 0.27 0.35 Total seed yield per plant (g) 0.74 0.09 0.51 0.28 Tuber fresh weight (g) 0.95 0.02 1.001 0.04 Tuber dry weight (g) 0.94 0.02 1.001 0.03 Seed harvest index (Sm) 0.66 0.13 0.39 0.23 Tuber harvest index (THI) 0.92 0.02 0.76 0.13 c. Phenological traits

Flowering (days after sowing) 0.86 0.03 0.70 0.21 First mature pod (days after sowing) 0.86 0.04 0.56 0.28 Seed development period (days after flowering) 0.68 0.13 1.001 0.03 I = estimate> 1.00; 2 = estimate < 0

119 There was some degree of dominance observed for some quantitative traits in the CPI 69030 x ACC 390 hybrid population, as indicated by the F! performance being relatively closer to one parent compared to the other parent. Simple ANOVA and Least Significant Difference (LSD) tests were perfonned to test the level of differences among the PI, P2, and F l means and to estimate whether the F1 performance for a specific trait was closer to the male parent (CPI 69030) or the female parent (ACC 390). Performance ofthe F] for tuber fresh weight was closer to CPI 69030, whilst for some traits, e.g. days to flowering, days to first mature pod, and duration of the seed development period, were closer to ACC 390. In contrast, there was no significant different between those three generations for tuber harvest index (Table 6.12).

Overall, most morphological traits exhibited high to very high broad sense and nalTOW sense heritability values, with the exception ofpod length and seeds per pod. The latter traits exhibited very small or zero nalTOW sense heritability values, while a moderate nalTOW sense heritability value was observed for flowers per peduncle (Table 6.13). High to very high nalTOW sense heritability values for some morphological traits, e.g. stem thickness, indicates that successful selection for those traits could be made in early generations (Lopes et a1.2003).

Even though the number of flowers per peduncle only exhibited a moderate nalTOW sense heritability value, very high nalTOW sense heritability values were observed for number of pods per feltile peduncle and the percentage of set pods (Table 6.13). A moderate nan-ow sense heritability value for number of pods per peduncle was repOlted by James and Lawn (1991) using different hybrid combinations of V. vexillata.

For pod length and width, very high broad sense heritability values were observed in this study (Table 6.13). Meanwhile, low nalTOW sense heritability was observed for pod length, in contrast to a very high nan-ow sense value for pod width. High broad sense heritability values in pod length have been also reported in cowpea (Omoigui et ai. 2006), and mungbean (Shanmugasundaram 1991), and moderate nan-ow sense heritability values for pod length were reported by Aryeetey and Laing (1973) and Bapna and Joshi (1973) in cowpea. Moderate narrow sense heritability was also reported for pod length in mungbean by Sriphadet et al. (2007). Leleji (1975) suggested short pod was partially dominant over long pod in cowpea.

Very high broad sense and narrow sense heritability values, 0.91 and 0.84 respectively, were repOlted by James and Lawn (1991) for the seed per pod trait. In the present study, a very high broad sense heritability value (0.99) was also observed, but the narrow sense heritability was estimated to be zero (Table 6.13). At least moderate broad sense heritability was reported for

120 seeds per pod in mungbean (Fery 1980). Even though both parents exhibited a large number of seeds per pod (>20 seeds), the F! plants had fewer than 10 seeds per pods (Table 6.12).

However, plants with large numbers ofseeds per pod were recovered in the F2 generation.

Very high broad and narrow sense heritability values were observed in these studies for 100 seed weight (Table 6.13). Fery (1980) suggested that seed size in both cowpea and mungbean was highly heritable under most environmental conditions. High broad and narrow sense heritability values for seed size were reported in cowpea by Drabo et al. (1984) and Lopes et al. (2003). Meanwhile, in V. vexillata, there was some variation in the estimates of broad and narrow sense heritability values rep011ed by James and Lawn (1991). In three hybrid combinations of V. vexillata, broad sense heritability values ranged from moderate to very high (0.45 - 0.86) while nalTOW sense heritability was low to very high (0.22 - 0.74). The large seed size of the CPI 69030 parent was not recovered in the F2 generation (Table 6.12). According to Leleji (1975), small seeds appeared to be partially dominant over large seeds in cowpea.

In terms of tuber diameter, CPI 69030 produced thicker tubers than ACC 390, whilst the F. plants were intermediate between the parents (Table 6.12). High broad and nalTOW sense heritability values were estimated for this trait (Table 6.13). High to very high broad sense heritability values were estimated for the main agronomic traits, but there was considerable variation in the estimates of narrow sense heritability, which ranged from low to very high (Table 6.13). For tuber fresh weight, tuber dry weight and TRI, very high values of narrow sense heritability were estimated from the population (Table 6.13). In contrast, low nalTOW sense heritability values were observed for TDM, whilst moderate to high narrow sense values were estimated for total seed yield and SHI. James and Lawn (1991) reported low to high broad sense heritability and low (zero value) to high nalTOW sense heritability for tuber attributes, seed yield attributes, and TDM.

The ability to mature early is a relatively important characteristic for many crop plants especially where water may be limiting (pery 1980; Adeyanju et at. 2007). The earliness trait is often measured as days to flowering or days to maturity (pery 1980). For ali the phenological traits in the present study, i.e. days to flowering, days to first mature pod, and seed development period, the performances ofthe F 1 plants was closer to ACC 390 (Table 6.12). High to very high values were estimated for both broad sense and narrow sense heritability (Table 6.13). In cowpea, Adeyanju et al. (2007) suggested that earliness, which was measured as days to first flowering, was controlled by dominant genes at two or more loci, whilst Brittingham (1950) cited by Fery (1980) postulated that maturity probably was inherited quantitatively.

121 6.3.4.5 Correlation between quantitative traits Overall, there were a large number ofconelations between pairs of quantitative traits that were observed in F2 generation in the CPI 69030 x ACC 390 hybrid population (Table 6.14). In further discussion, the ternl "highly significant" refers to con-elation coefficients significant at P ~ 0.01, whilst the term "significant" refers to significance at P < 0.05.

In relation to yield per plant, some morphological and agronomic traits had highly significant correlations with both seed and tuber yield. For instance, stem thickness had positive conelations with pod width (r = 0.339) and 100 seed weight (r = 0.411), whilst number of pod per fertile peduncle had positive correlations with total seed yield per plant (r = 0.325) and SRI (r = 0.279). Even though the number of pods per peduncle had highly significant correlation with total seed yield per plant, in lentil Tambal et al. (2000) recommended avoiding early selection by number ofpods per peduncle because it was not automatic that the plant which the had highest number ofpods per peduncle also exhibited the highest yield.

Pod length in legumes was often reported to be associated with the number of seeds per pod (Grant et al. 2003; Lawn and Holland 2003; Biradar et al. 2007, Makeen et al. 2007), whilst pod width was associated with seed size (Lawn and Holland 2003; Dursun 2007). This was also observed in this study, where pod length had high positive correlation with seed per pod (r = 0.780), whilst pod width had a positive cOlTelation with 100 seed weight (r = 0.827). Pod length also had positive correlations with total seed yield per plant (r = 0.600) and SHI (r = 0.480) (Table 6.14). Meanwhile, total seed yield had high correlations with all agronomic traits except TDM. Mostly, it had high negative correlations with all tuber attributes, i.e. tuber diameter (r = -0.383), tuber fresh weight (r = -0.373) and tuber dry weight (r = -0.352), and to THI (1' = ­ 0.398) (Table 6.14). It suggested that the plants exhibiting higher total seed yield per plant tended to allocate photosynthetic assimilate for seed production rather than store it in the tuber.

There were high correlations observed within tuber attributes and the traits that related to them, e.g. tuber diameter had high positive cOlTelations with tuber fresh weight (r = 0.765), tuber dry weight (r = 0.739), TDM (r = 0.485) and THI (r = 0.704). Tuber fresh weight had high positive correlations with tuber dry weight (r = 0.975), TDM (r = 0.605), and THI (r = 0.873), whilst a negative correlation was observed with SHI (r = -0.593) (Table 6.14).

122 Table 6.14 Linear correlation co-efficients among pairs ofquantitative traits observed in the CPI 69030 x ACC 390 hybrid population.

Traits§ ST TLL TLW LWR FP PP pSP PL PW SP WS TD TOM SY FWT OWT 8Hl F FMP SDP ST 1.00 TLL 0.29" 1.00 TLW 0.43" -0.38" 1.00 LWR -0.26" 0.55" -0.85" 1.00 FP 0.08 -0.09 0.G7 -0.12 1.00 PP -0.15 -0.02 -0.08 0.04 0.60" 1.00 PSP -0.24' 0.06 -0.17 0.17 -0.23' 0.63" 1.00 PL 0.02 0.17 .0.17 0.G7 -0.01 0.12 0.16 1.00 PW 0.34'- 0.32" 0.04 -0.01 -0.02 -0.10 -0.12 -0.D3 1.00 SP -0.20' 0.02 -0.29-' 0.16 O.Ol 0.15 0.17 0.78" -0.28-- 1.00 I WS 0.41" 0.23' 0.12 -0.08 0.01 -0.12 -0.17 -0.13 0.83-' -0.44" 1.00 TO 0.22' -0.15 0.39'" -0.36" 0.04 -0.13 -0.19 0.33" 0.19 -0.56" 0.35" 1.00 TOM 0.25' 0.04 0.29'" -0.18 0.01 -0.19 -0.22' -0.16 0.18 -0.36" 0.34" 0.48" 1.00 SY -0.02 0.27" -0.23- 0.16 0.18 0.32" 0.21' 0.60" 0.12 0.60" 0.04 -0.38" -0.16 1.00 I FWT 0.20' -0.14 0.45'" -0.40" 0.02 -0.12 -0.14 -0.19 0.05 -0.48" 0.20' 0.76" 0.60'· -0.32" 1.00 DWT 0.16 -0.11 0.39" -0.36·· -0.D3 -0.13 -0.11 -0.17 0.08 -0.46" 0.21' 0.74" 0.67'- -0.35" 0.97" 1.00 8m -0.15 0.15 -0.32-- 0.22' 0.09 0.28" 0.24- 0.48" -0.03 0.61·' -0.23· -0.52'- -0.76" 0.70" -0.59" -0.63" 1.00 I F 0.06 -0.05 0.22' -0.09 -0.14 -0.29" -0.20' -0.25' -0.16 -0.10 -0.l4 0.02 0.28" -0.29" 0.03 0.05 -0.37" 1.00 FMP 0.11 -0.09 0.26' -0.14 -0.10 -0.32" -0.27" -0.35** -0.11 -0.21' -0.06 O.ll 0.32" -0.35" 0.08 0.08 -0.43" 0.96·· 1.00 I SOP 0.19 -0.12 0.11 -0.15 0.14 -0.08 -0.21' -0.32" 0.18 0.36'· 0.31" 0.28" 0.11 -0.16 0.16 0.11 -0.17 -0.25' 0.03 I 1.00 § ST, stem thickness; TLL. terminal lcaflet length; TLW, terminallcatlet width; LWR, length/width ratio; FP, numbcr oftlowers per pedunclc; PP, number ofpods per peduncle; PSP, percentage ofsetting pod; PL. pod length; PW, pod width; SP, number ofseeds per pod; WS, weight of 100 seeds; TD, tuber diameter; TDM, total dry matter; SY. total seed yield per plant; FWT, fresh \Veight of tuber; DWT, dry weight of tuber; SHI. seed harvest index; F, time 10 flowering; FMP. time to first mature pod; and SDP, seed development period. " ** significant at P < 0.05. 0.0 I respectively

123 Within the phenology traits, time to flowering had a strong positive correlation with time to first mature pod (r = 0.961), whilst a low but significant negative correlation was observed between flowering and seed development period (r = -0.246) (Table 6.14). In relation to agronomic traits, times to flowering and to first mature of pod had highly significant negative correlations with total seed yield per plant (r = -0.295 and r = -0.350, respectively), and consequently with SHI (Table 6.12).

6.3.6 Summary and conclusions The original idea to explore the inheritance of traits using hybrids involving the cultivated Bali accessions was prevented by the incompatibility ofthese accessions with var. macrosperma CPI

69030 and many wild types, and the infertility of the F2 hybrids between the Jim lines and the Australian wild accession ACC 390. In contrast, a lot of information was collected using the hybrids between the var. macrosperma x wild type accessions. In general, the cultivated traits in the latter populations were mainly controlled by single recessive genes, with the wild type traits dominant. To the limited extent that comparative data were available from the Bali x wild hyblids, similar responses were observed. Interestingly, the broad leaflet shape of both the Bali accessions and var. macrosperma was controlled by a single recessive gene - which contrasted with broad leaflet shape in wild African accessions (James and Lawn 1991). The fact that many of the wild type qualitative traits were dominant means that they could be fairly easily eliminated from breeding populations, because selection for the cultivated type would recover homozygous recessive genotypes and so exclude the wild genes.

In general, broad sense heritabilities were high for most morphological, yield-related and phenological traits observed in the var. macrosperma x wild type hybrid populations. Narrow­ sense heritabilities were also moderate to high for most traits, except for some pod traits (seed per pod and pod length) and phenology traits, especially time to flowering. These responses suggested that in these populations at least, many ofthe quantitative traits ofinterest to the plant breeder were under largely additive genetic control, and therefore would be responsive to selection. However, caution is needed in applying these findings to populations involving more closely-related parents e.g. populations involving only cultivated accessions.

The success in achieving some backcrosses using the infertile F I hybrid between the cultivated Bali and wild accessions suggested it may be possible to expand genetic variability in the cultivated populations using these wild types, but the process would be slow and laborious. Probably, it would be more worthwhile to try to find wider diversity among the Bali types before using this backcrossing approach.

124 7.0 GENERAL CONCLUSIONS AND IMPLICATIONS

\Xlll" ll" 1111 Tn_ thp.a_ introrhwt;o!1- --_ to thi" thp"i", V.. 1JPrillntn.. ~., _...... - hieahliahtpt1o _- - -_ llnnpY-lltili"F'n__ _- IF'O'llmp0 ... species that has several multi-purpose attributes. Its high-protein seeds, pods and tubers make the species a potentially valuable food crop in its own right for improving village agriculture in the tropics. In addition, its close relations to both the African and Asiatic Vigna species make it a potentially useful source of traits for improving other major pulse crops within Vigna. Since prior to this study, there was only limited information on domesticated types within this species, it was considered imp0l1ant to explore the areas where additional knowledge would help underpin future improvement research and varietal development in V. vexillata.

The broad aims of the work reported here were to observe and document (a) the genetic diversity based on morphological, agronomic and phenological traits within V. vexillata accessions that had been collected from Africa, Australia and Indonesia; (b) the genetic compatibility among them based on hybridisation studies; and (c) the inheritance of selected qualitative and quantitative traits, with emphasis on comparing cultivated and wild traits. Obtaining comprehensive information in those key areas was seen as the first priority for developing future breeding programs for V. vexillata, as outlined below.

The knowledge ofgenotypic variation within a species is a basic tool for breeders to be able to improve particular traits. Sufficient genotypic variation is needed for the process of varietal development. Therefore, the first key area which was identified to improve V. vexillata was to explore the genetic variability of several key traits, both qualitative and quantitative, among the available three classes of accessions: the cultivated Bali accessions, the large-seeded var. macrosperma which seemed to share some ofthe cultivated traits and wild accessions from both Austronesia and Africa.

InfOlmation on the genetic compatibility within a species is important as a base of further improvement of that species, especially when the breeder needs to improve genetic variability within the species or to transfer one or more important traits from one plant to another through hybridisation. Therefore, the second key area which was investigated to support V. vexillata improvement was exploring the genetic compatibility among the three main classes of accessIOn.

Finally, knowledge ofthe inheritance / heritability ofqualitative and quantitative traits is needed to provide guidance to the breeder when seeking to improve particular traits. Therefore, the third

125 key area which was identified was to investigate the inheritance of qualitative and quantitative traits, particularly domesticated vs. wild traits, in V vexillata.

7.1 Key outcomes There were several outcomes from the experiments described herein. Firstly, it was established that there was large phenotypic variation between the three main classes ofaccessions that were evaluated, namely, the cultivated Bali accessions, var. macrosperma and the wild accessions. In addition, there was significant vatiation for most traits among the wild accessions from both Africa and Austronesia. There was almost no genotypic variation observed within the several cultivated Bali accessions. Unfortunately, only one unique accession of var. macrosperma was available for study.

Secondly, there were various levels of genetic incompatibility between the three classes of accessions. While the wild accessions and var. macrosperma were inter-fertile and hybridized easily, incompatibilities were observed between the cultivated Bali accessions and the two other classes of accession. It was confirmed that there were no differences in chromosome number within the selected accessions which were subjected to mitotic analysis.

Thirdly, a study of the variation in hybrid progeny confirmed that the inheritance or heritability of a range of qualitative and quantitative traits were broadly consistent with previous reports both within V vexillata and for other legume species. Consistent with previous studies, inheritance of some of the observed wild qualitative traits was apparently controlled by single dominant genes.

The bases for these conclusions are discussed briefly in tum below.

7.1.1 Genetic diversity for morphological, agronomic and phenological traits Based on the traits observed, it appeared that the largest variation in both qualitative and quantitative traits occurred between the cultivated Bali accessions and var. macrosperma on the one hand and the wild accessions on the other. The cultivated Bali accessions and var. macrosperma were distinguished by traits often associated with domestication whereas the wild types were characterized by traits that appeared to link to their adaptation within natural environments. For instance, the first two classes ofaccession exhibited large harvest (pod, seed) and non-harvest (leaflet and tuber size, stem thickness) organs, light-coloured seeds, high seed yield per plant and non dehiscent pods. In contrast, the wild types were characterized by small, dark seeds, strongly dehiscent pods, fine twining stems, and in most cases, small leaflets.

126 Despite the many similarities between the Bali accessions and var. macrosperma, there were differences. The Bali accessions exhibited much greater number of flowers per peduncle and number of pods per peduncle compared to vaL maCrO:5perma which was closer to the wild accessions for this trait. The Bali accessions also had fewer seeds per pod. One specific trait, which is also often associated with domestication, namely robust growth habit, was only exhibited by var. macrospenna.

Based on the phenological traits observed, particularly time to flowering, it was clear that the Bali accessions exhibited strong photoperiod sensitivity which distinguished those accessions from var. macrosperma. The pattern ofresponse in time to flowering in the Bali accessions was consistent with quantitative short-day photoperiodic adaptation, where the longer photoperiods appeared to significantly prolong the vegetative growth period and delay flowering. Despite their having been reported as cultivated, the strong photoperiod sensitivity exhibited by the Bali lines suggested that they have been only partially domesticated.

Notwithstanding the fact that the Bali accessions are the only germplasm cUlTently located in international collections that is unequivocally described as being "cultivated", val'. macrosperma demonstrated several traits that suggested it was at least as domesticated as the Bali lines, ifnot more so. As well as sharing many traits that are favoured during domestication, e.g. larger seed size and larger non-harvest organs with the cultivated Bali accessions, the var. macrosperma accession was characterized by very robust, almost bush-like growth habit, in contrast to the twining, viny habit of the Bali accessions. Moreover, at least in Townsville, the var. macrosperma accession was largely insensitive to photoperiod. Perhaps as a result, pod fonnation in the var. macrosperma accession was relatively unifonn, with most pods maturing over a short time. The original provenance ofTVNu64 as CPI 15452 from legume introduction and evaluation trials in The Sudan support the conclusion that it is in fact a domesticate.

7.1.2 Genetic compatibility among cultivated Bali, var. macrospemza and wild accessions Given that the previous study of genetic diversity revealed that there was very low genetic variability within the cultivated Bali accessions, the ability to introduce variability through hybridization with accessions from the other two classes assumed greater importance. Therefore, this study was a very impol1ant step in determining the scope to improve genetic variability of V. vexillata particularly within those accessions.

Despite the confounding effects of high temperature and long photoperiod, which appeared to influence the rates of success of some of the artificial hybridisations, the technique was sufficient to obtain successful hybrids from one or more different hybrid combinations between

127 the three classes of accession. The results demonstrated there were clear underlying differences in genetic compatibility between accessions from the three different V. vexillata classes. There was no evidence of genetic breakdown when var. macrosperma was hybridised with wild accessions from Austronesia or Africa. In contrast, the hybridisations between the cultivated Bali accessions and accessions from the other two classes all demonstrated hybrid breakdown. Breakdown mechanisms were observed at one or more stages of development in those hybrid combinations: pollinated flowers abscised, pods set but abscised prematurely, pods matured but hyblid seed were shrivelled, hybrid seed appeared normal but failed to germinate, hybrid seed germinated but young hybrid seedlings died, and/or hybrid plants were vigorous but sterile. Despite the morphological similarities between the cultivated Bali accessions and the var. macrosperma accession, it was clear that genetic barriers prevented easy hybridisation between them. The responses observed here suggest that the cultivated Bali accessions are genetically distinct from the wild African, wild Austronesian and var. macrosperma accessions which in tum are closely related.

In order to identify the possible cause of the hybrid breakdown, mitotic analysis was conducted for the cultivated Bali x wild Austronesian hybrids to examine chromosome numbers during mitosis in the parents and hybrids. While genetic breakdown may occur in hybrids between parents with the same number of chromosomes, as has been reported in rice (Li et af. 1997; Kubo and Yoshimura 2002; Yamamoto et af. 2007), differences in chromosome number between the two parents can be a cause of hybrid breakdown. Due to difficulties in obtaining meiotic chromosome counts, only mitotic analysis was able to be pelformed. Based on these analyses, it was evident that the cultivated Bali accessions, the wild Austronesian accessions and their hybrids all exhibited the same number ofchromosomes (2n = 22).

Pollen viability analyses using Alexander's stain were also conducted to investigate the possible cause ofthe observed sterility ofthe Bali x Australian hybrids. The percentages ofviable pollen and the numbers of pollen grains in the hybrids were significantly lower than for both the cultivated and wild parent accessions. Even though the validity of pollen viability analysis has been subject of discussion (Dafni and Firmage 2000), it does yield comparative data and the results presented in this study suggest that genetic breakdown which occurred in the hybrids between the cultivated Bali and the wild accessions of V. vexillata were influenced by pollen development which was expressed in terms of both impaired pollen production and reduced pollen viability. Consistent with this observation, a small number of viable seeds was obtained when viable pollen from the parents was backcrossed onto the sterile hybrids.

128 7.1.3 Inheritance of putative wild and domesticated traits Some of the hybrids developed in the genetic compatibility studies were used to explore the inheritance of wild and domesticated traits. In this research, three subsets of experiments were conducted, each involving hybrids between a wild parent as one parent and either var. macrosperma or a Bali cultivated accession as the other. In the event, because of the infertility ofthe Bali x wild hybrids, only limited observations were possible with those populations.

The main inheritance study involved a complete population set (Ph P2, FI, BCPI, BCP2, F2) from the var. macrosperma x wild Australian hybrid. Plants from each population for this hybrid were grown to enable a comprehensive inheritance of selected putative wild and domesticated traits. In this experiment, it was evident that lanceolate leaflet shape and narrow leaflet width, both of which were characteristic of the Australian wild parent, were each controlled by single dominant genes. Other traits that are also often associated with wild plants, e.g. black-speckled seed testa pattern and dehiscent pods, were also controlled by single dominant genes.

For most of the quantitative traits measured, the F] generation exhibited mid-parent values, suggesting largely additive genetic variance. Some attributes which are often associated with domestication, e.g. seed size and stem thickness, exhibited high narrow sense heritability suggesting they would be easy to breed for. Likewise, tuber traits tended to have high narrow sense heritability. However, narrow sense heritability for some important agronomic traits, like seed yield per plant and harvest index, were only low to moderate, suggesting that those traits would be difficult to breed for compared to seed size, stem thickness and tuber traits.

Limited observations on trait inheritance were also made on the two backcross populations of the cultivated Bali (Jim IA and Jim 2) x wild Austronesian (ACC 390) accessions. These observations were consistent with lanceolate leaflet shape and narrow leaflet width being controlled by single dominant genes, as for the var. macrosperma x wild hybrid discussed above. Meanwhile, time to flowering appeared to be controlled by several genes, due to the fact that the F I hybrid plants and the backcrosses to the wild parent all flowered later than the wild parent, but most flowered sooner than the cultivated parent. In the backcrosses to the cultivated Bali accessions, it was apparent that those backcross plants were strongly photoperiod-sensitive like the cultivated parents, as few flowers were produced during the evaluation, which of necessity was sown in the early summer.

In these cultivated x wild backcross populations, it was observed that the backcross plants, like the F 1 hybrids, were generally infertile. Flowers developed but generally no seed was obtained

129 even when 'tripping' was performed. In at least one instance, however, seeds were obtained in the backcrosses to the wild Australian parent when 'tripping' was performed. The formation of some seeds in some of the backcross plants indicated SOlue recovery of feitility, suggesting that it may be possible to transfer traits between the cultivated Bali and wild populations.

A third subset of experiments involved limited observations of leaflet traits in two hybrid populations involving var. macrosperma x wild African accessions. Observations were made only on the parent accessions and their F I hybrids. As for the two previous sets of populations, the inheritance of leaflet shape and width was consistent with single dominant gene control. It was of interest that the inheritance of the qualitative leaflet traits common to both the Bali and var. macrosperma accessions, namely, leaflet shape and width, was similar, even though it was evident from the genetic compatibility study (section 7.1.2) that the two classes were genetically distinct. For both accession classes, ovate leaflet shape and wide leaflets were recessive to the wild lanceolate leaflet shape and narrow leaflets.

7.2 Implications of the studies Based on the key outcomes as outline above, there were several key implications ofthe studies as described below.

The most important of these is the consequences for implementing a breeding program to develop improved varieties of cultivated V vexillata. As described in section 7.1.1, the genetic variability observed in the cultivated Bali accessions was almost non-existent. Certainly, it was almost impossible to visually distinguish between the hybrids between the accessions from Jimbaran and Tabanan regions and their parents. Because genotypic variability is one of the main factors required to be able to implement a successful breeding program, it would be very difficult to base a breeding program to develop cultivated varieties of V vexillata solely on the cultivated Bali accessions currently available. It would be necessary therefore to explore ways of expanding the available genetic variability in order to develop of improved varieties of V vexillata using the cultivated Bali type.

One possibility would be to undertake a comprehensive collection program on Bali to try to find greater genetic diversity. However, given that the accessions used here were collected from two different regions, it is possible that only limited additional diversity would be found. Altematively, attempts could be made to source additional germplasm from other countries. For example, it is possible that types similar to the Bali accessions exist in Sri Lanka (Remy Pasquet, email communication to RJ Lawn, 2008). Given the genetic incompatibility between the Bali accessions and the other classes of V vexillata accession, particularly var.

130 macrosperma, it is clear that these could only be used as sources of specific traits important enough to justify the effort ofmoving them across.

Molecular markers may identify greater variability within the cultivated Bali accessions at individual gene level than was apparent at the whole plant level in these studies. For example, while it was difficult to distinguish between the Jim and Tab lines for many traits, there were distinct differences in their compatibility with wild accessions. Viable hybrids were achieved with wild accessions using the Jimbaran accessions whereas the Tabanan x wild hybrids were completely non-viable. The development and use of biochemical-based techniques and molecular marker technologies for genetic variability assessment, such as restriction fragment length polymorphism (RFLPs), amplified fragment length polymorphisms (AFLP), random amplified polymorphic DNA (RAPDs), and simple sequence repeats (SSRs), have been established in several important crop plants (Grivet and Noyer 2003). For instance, molecular markers have been used to study the genetic relationships among species in the genus Vigna (Ajibade et al. 2000; Tomooka et al. 2002; Saravanakumar et al. 2004; Seehalak et al. 2006) and to analyse diversity within Vigna angularis (Yee et al. 1999; Mimura et al. 2000; Xu et al. 2000), V. unguiculata (Coulibaly et al. 2002; Fang et al. 2006; Ba et al. 2004); and V. radiata (Lakhanpaul et al. 2000). Molecular markers also have been used to analyse diversity within Vigna vexillata (Spinosa et al. 1998).

Alternatively to the cultivated Bali accessions, val'. macrosperma might be used as the basis for breeding cultivated varieties of V. vexillata. However, again there appears to be limited variation available, given the very few accessions in international collections (as described in Section 2.3). As for the Bali type, it is clear that a widening of the gene pool of var. macrosperma accessions is urgently needed before a breeding program to improve cultivated V. vexillata can be implemented. In contrast with the Bali types, wild accessions could be used to broaden the gene pool available with val'. macrosperma. However, this would complicate progress by introducing many wild type genes.

The basis of the genetic incompatibility between the Bali accessions and the other accessions remains unclear. In the absence of data £i:om the meiotic analysis, the pollen viability and mitotic analyses did not permit specification of the mechanism of genetic breakdown between the cultivated Bali and the wild accessions. Therefore, future meiotic analysis is necessary to further investigate the genetic breakdown mechanism which occurred in the hybridisation between the cultivated Bali and wild accessions. Using meiotic analysis, possible causes of genetic breakdown could be confirmed, for example, irregular chromosomal pairing and subsequent rearrangement during meiosis, which often causes sterility in the hybrid between

131 two distantly related species (Ladizinsky 1998; Rieseberg and Carney 1998) or other factors, such as the interaction of two recessive genes as reported in rice (Li et al. 1997; Kubo and

Yoshimura 2002; Yamamoto et al. 2007), which was confirmed with SUppOlt of DNA marker analysis (Li et al. 1997; Kubo and Yoshimura 2002). Thus the use of molecular markers could also be infolmative in attempts to further elucidate the mechanism of genetic breakdown in V. vexillata.

The last important implication of these studies relates to the taxonomy of the V vexillata complex. From the genetic compatibility investigations (see Chapter 4), it was evident that the cultivated Bali accessions have strong genetic incompatibility with both the wild accessions of V vexillata and with var. macrosperma, notwithstanding some morphological similarities between the var. macrosperma and the cultivated Bali accessions. Based on the gene pool concepts of Harlan and Wet (1970), that condition suggested that the cultivated Bali accessions do not belong to the same gene pool as the wild accessions or even with val. macrosperma, despite their shared morphological similarities palticularly for attributes often associated with domestication. Therefore, a taxonomic revision is required for the cultivated Bali accessions in order to confirm their taxonomic status.

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