Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2017
Volume 20
Issue 4
Topic:
Agronomy
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Galek R. , Kozak B. , Sawicka-Sienkiewicz E. , Zalewski D. , Nowosad K. 2017. SEARCHING FOR THE MOST USEFUL GENOTYPES OF LUPINUS MUTABILIS SWEET FOR BREEDING PURPOSE, EJPAU 20(4), #11.
Available Online: http://www.ejpau.media.pl/volume20/issue4/art-11.html

SEARCHING FOR THE MOST USEFUL GENOTYPES OF LUPINUS MUTABILIS SWEET FOR BREEDING PURPOSE

Renata Galek, Bartosz Kozak, Ewa Sawicka-Sienkiewicz, Dariusz Zalewski, Kamila Nowosad
Department of Genetics, Plant Breeding and Seed Production, Wroc³aw University of Environmental and Life Sciences, Poland

 

ABSTRACT

Knowing the diversity of traits is basic for elaborating selection strategy in breeding new crop - Lupinus mutabilis in moderate climatic condition.  Multivariate methods (PCA and CA) were applied for proper evaluation twelve genotypes in respect to 16 morphological traits, yield structure and DNA polymorphism using eight SSR and eleven ISSR markers. The first three principal components accounted for 77.53% of the overall variability. The first component  was connected with: the height of main stem, whorls and  flowers numbers on the main stem, as well as pods number from lateral branches.  Molecular markers generated of 121 polymorphic amplification products. The CA performed on the basis of morphological traits, yield structure and DNA markers showed the different content of grouped genotypes. This fact indicates a need to extend molecular analysis with other types of markers, useful in selection of genotypes desirable from the breeding point of view.

Key words: ISSR, Lupinus mutabilis, multivariate analysis, SSR .

INTRODUCTION

The genus Lupinus constitutes a large group of interesting and, simultaneously, useful species which have not fully been taken advantage of in spite of many ages’ tillage tradition. Only the annual species, namely the yellow, white, narrow-leaved and Andean lupin, have been used in agriculture. The last species mentioned, called the Andean soya bean thanks to its high content of proteins (up to 50%) and fat (20%), makes a valuable component of human diet and an important nutrient in animal feeding. It is grown in South America at altitudes ranging from 1800 to 3000 m a.s.l. [3, 26]. From the animal breeding and agricultural perspectives, the advantages of the Andean lupin include a soft and thin seed coat, non-shattering and indehiscent pods (characters which are of particular importance in the case of mechanic harvest), and also the plant does not display photoperiodic response. These traits are a result of selection pursued by Andean farmers. So far, no evidence has been found to confirm that the process of domestication of L. mutabilis was unreiterated [11, 26]. Among its essential disadvantages, high alkaloid content in seeds and sensitivity to anthracnose should be mentioned [12, 13, 26]. However, the bitter forms should not be eliminated from agriculture as the Andean lupin seed extract has been found to exert a positive influence in cases of glucose deficiency (hypoglycaemia) in treatment of diabetes. The genistein, contained in bitter seeds, is used in the production of nutraceutics for diabetic patients [2].

The process of acclimatization of the Andean lupin to the conditions of moderate climate was launched in the 90s of the last century - project was realized in international cooperation (AIR3-CT93-0865). In parallel, attempt were started to induce mutants, and to search for and select genotypes which would possess desirable traits, including, most of all, forms characterized by a shorter vegetation season period, thinner seed coat and reduced development of lateral branches, reduce the alkaloid content [3, 14, 27].

Attempts at acclimatization of the species have also recently been undertaken in western Australia [1, 6, 7, 25]. Cultivation of the Andean lupin is also pursued at the Agropecuaria National Research Autonomous Institution (INIAP) in Ecuador, where ca 120 taxa have been gathered. The activity has resulted in acquisition of a few cultivars adapted to the local conditions [13].

Considering the growing interest in the Andean lupin in different parts of the globe, it is necessary to intensify the breeding activity and to recognize the variability of its agronomic traits under local conditions. In connection with this, it is indispensable to more extensively employ various statistical models – based not only on univariate but also on multivariate methods – for assessment of breeding material [15, 18, 22]. Multivariate methods enable a breeder to determine the structure of variability regarding pertinent properties. A significant aspect of a breeding strategy is also the knowledge of the polymorphism of the initial material at the level of DNA [5, 24].

The aim of the undertaken study was to conduct a multivariate analysis of variability with regard to sixteen simultaneously analyzed traits and DNA polymorphism of the collected L. mutabilis material.

MATERIAL AND METHODS

The experimental material
The experimental material consisted of twelve L. mutabilis genotypes (XM.5, LM.13, LM.34, Mut-45, Mut-220, 1H_XM.5 x KW, 2H_XM. x KW, 5H_LM.13 x KW, 6H_Mut.160 x KW, 7H_Mut.220 x KW, 3H_LM.34 x Mut.45, 4H_LM.34 x Mut.45). Three years’ field experiments were set up by the randomized block method in three replications. Estimation of 16 morphological traits and yield components was performed: main stem height [cm], plant height (cm), number of lateral branches, inflorescence length of main stem [cm], whorls number on the main stem, number of  flowers per the main stem, pods number on the main stem, harvest index of the main stem, inflorescence length of the lateral branches [cm], whorls number on the lateral branches, number of  flowers per lateral stem, number of pods  from branches, harvest index on lateral branches, weight of 1000 seeds main stem [g], pods number from lateral branches, number of  pods from plant. Measurements were made on 10 randomly selected plants per plot.

Statistical analysis – morphological and yield structure characters
For grouping objects in respect of phenotypic similarity, a cluster analysis (CA), with application of the Ward method, was carried out (Legendre and Legendre, 2012). To detect relationships between the investigated traits, analyses of multidimensional data were conducted – cluster analysis and principal component analysis (PCA). For calculations, the R Software [20] was employed. Arithmetic means for the three-year period underwent standardization before being subjected to statistical analysis.

DNA polymorphism characteristics
Eight pairs of SSR (Simple Sequence Repeats - LSSR05, LSSR06, LSSR07, LSSR09, LSSR10, LSSR11, LSSR14, LSSR41) primers and eleven pairs of ISSR (Inter Simple Sequence Repeats - UBC807, UBC808, UBC809, UBC814, UBC815, UBC816, UBC817, UBC821, UBC822, UBC823, UBC826, UBC827, UBC828, UBC829, UBC842) primers (set # 9 University of British Columbia) specific to Lupinus ssp. were used to determine the DNA polymorphism [23]. Genomic DNA was extracted from young leaves by the CTAB method [9] with minor modifications as described by Clements et al. [8].

Amplification of DNA for ISSR markers was performed in 15 µl reaction mixture containing 10 mM Tris-HCl, 50mM KCl, 0.08 % (v/v) Nonidet-P40, 2 mM MgCl2, 0.2 of each dNTP, 0.4 µM primer, and 1 U of Taq DNA polymerase enzyme (Fermentas, Canada) and 45 ng of DNA using a Biometra thermal cycler for 40 cycles. After initial 4 min denaturation at 94 °C, each following cycle comprised of 1 min denaturation process at 94 °C, 45 s annealing at 50 °C, 2 min extension at 72 °C with a final 5 min extension at 72 °C at the end of 40 cycles. Amplified products were then separated by electrophoresis in 2.0% agarose gels and visualized with ethidium bromide (1.0 μg*ml-1). The electrophoretic patterns of the PCR products were photographed under UV light. Analysis were performed in two replications.

Amplification of DNA for SSRmarkerswas performed in a 15-µl reaction mixture, using a 2×PCR mixture that contained Taq polymerase 0.1 U/µl, dNTP mix 2 mM, MgCl2 4 mM (AA Biotechnology, Poland), 0.28 µM of each primer and 45 ng of DNA, using the Biometra thermal cycler for 35 cycles. After initial denaturation for 5 min at 94°C, each cycle was composed of 30-sec denaturation at 94°C, 30-sec annealing at 55°C, 30-sec extension at 72°C with a final extension for 7 min at 72°C at the end of the 35th cycle. The amplified products were analyzed with the application of the QIAxcel (Qiagen, Germany), using the QIAxcel DNA screening Kit (Qiagen, Germany) and according to the AM420 method. The QX Alignment Marker 15 bp/3 kbp (Qiagen, Germany) and QX DNA.Size Marker 100 bp–2.5 kbp (Qiagen, Germany) were employed in the analysis.

Molecular data analysis
Each fragment that had been amplified was treated as a unit character and scored in terms of binary code (1/0=+/-). The data were entered in the MS Excel spreadsheet to create a binary matrix. A matrix of the Nei genetic identity (Is) and genetic distance (Ds=1-Is) was calculated according to the formula Is= 2Nij/(Ni+Nj), where Nij is the number of bands present in both genotypes i and j, Ni is the number of bands present in genotype i, and Nj is the number of bands present in genotype j [19].

For objects grouping based on the genetic distances, the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) was used. The dendrogram was created with the R software [20] based on the Nei genetic distance matrix.

RESULTS AND DISCUSSION

A univariate analysis has revealed significance of differences for 12 genotypes in regard of all traits. After the conducted multivariate analysis, a conclusion was drawn that the first three principal components explained 77.53% of the total multivariate variation of all the studied means of traits (Table 1). The first variable accounted for 37.15% of the variation among the tested materials.  Such features as: inflorescence length of the main stem, whorls number of the main stem, number of flowers per the main stem and pods number from lateral branches became the most strongly and clearly differentiated within the tested material (Table 1). The second main component explained 24.18% of variation in the analyzed genotypes. The largest shares in the formation of this component were those of the whorls and pods number on the lateral branches. The harvest index of the main stem was the most correlated trait with the third principal component. This variable accounted for 16.20% of the variance (Table 1).

Table 1. Eigenvectors of the 16 pheno-morphological and agronomic traits for first third components (PCs) in 12 genotypes of Lupinus mutabilis
PC1 (37.15%)
PC2(24.18%)
PC3(16.20%)
Traits
Eigen vector
Main stem height [cm]
0.67
0.25
0.62
Plant height [cm]
0.44
0.56
0.38
Number of lateral branches
-0.35
0.49
0.31
Inflorescence length of main stem [cm]
0.82
-0.10
0.46
Whorls number on the main stem
0.85
-0.44
0.05
Number of  flowers per the main stem
0.88
-0.35
0.08
Pods number on the main stem
0.18
-0.63
0.64
Harvest index of the main stem
-0.38
-0.36
0.75
Inflorescence length of the lateral branches [cm]
0.65
-0.22
0.02
Whorls number on the lateral branches
0.54
-0.73
-0.21
Number of  flowers per lateral stem
0.48
-0.67
-0.32
Number of pods  from branches
-0.50
-0.77
0.02
Harvest index on lateral branches
-0.72
-0.39
0.23
Weight of 1000 seeds main stem [g]
0.23
0.55
0.55
Pods number from lateral branches
-0.81
-0.29
0.23
Number of  pods from plant
-0.64
-0.52
0.47
Note. Values in bold indicate values>0.7 for each principal components

Selection of the best genotypes with regards to yield structure traits causes numerous difficulties. Therefore, the most suitable are multivariate analyses. They allow efficiently examining the relationships between many investigated variables, as well as contributing to better understanding of the traits structure in different plant species collections [15, 17, 22, 27]. Features that make up the first, second and subsequent components of the main variability have the strongest discriminatory effect among the tested genotypes. In the case of Lupinus mutabilis there is a lack of work aimed at evaluation of multivariate analysis in the choice of the best genotypes for further selection. The herein discussed research represents one of the first attempts at describing the material gathered in respect of agronomically useful characters. Our results concerning L. mutabilis show that three components explain almost of the whole variability (Table 1).

In the research by Lema et al. [17], who pursued an analysis of principal components for the narrow-leaved lupin, the first three principal components were established to account for 66% of the overall multi-trait diversity. The greatest contribution (41%) in the formation of the first component was that of characters pertaining to seeds (length, breadth, weight), and also ones linked with water permeability through the seed coat and with early start of the blooming season.

Zalewski et al. [27] reported about multivariate analysis used for evaluation of wild and cultivated lupin species. The first three principal components explained 75.8% of the main variability. The first principal component was found to account for 38.4% of the variability. The phenological phases and the height of a plant had the greatest contribution to this particular component. Similarly as in the presented research, the number of pods from lateral stems had the greatest share in the second principal component in wild and cultivated species of Lupinus [27]. The other investigated characters did not play the same role in the main variability in both cases of the experimental series.

A cluster analysis showed diversity of the studied genotypes for sixteen screened traits (Figure 1). Twelve genotypes were grouped into seven clusters similar in respect of analyzed traits. The first cluster was represented by two genotypes. These lines were characterized by high plants, a significant number of flowers, long inflorescences of the main stem and a relatively high 1000-seed weight. Two lines, i.e. 1H_XM.5 x KW and XM.5, were located in a second cluster, and they were characterized by unfavorable characters, which is fewer pods and a lower harvest index both for the main stem and for the lateral branches. In addition, these lines had a low weight of 1000 seeds and a limited number of lateral branches. Line no. Mut-220 formed a separate group, which was characterized by favorable traits such as a limited number of lateral branches, and numerous flowers and pods set on the main stem. The genotypes assigned to the fourth (2H_XM.5 x KW 4H_LM-34 x Mut-45) and the fifth group (5H_LM.13 x KW 6H_Mut-160 x KW) differed mainly in the mean value of 1000-seed weight, but approximated to the other genotypes in the remaining traits. The mean values of the traits of line no. Mut-45 significantly differed from the average total. The plants were lower, with shorter inflorescences, fewer whorls and flowers on the main stem. They had desirable features from a breeder’s point of view – a higher average number of pods and were distinguishable by a harvest index higher than the average. In addition, this line had a lower weight of 1000 seeds (Table 2). Lines LM.34 and 3H_LM.34 x Mut-45 formed more lateral branches in comparison with the other genotypes. The remaining characters did not diverge from the overall mean.

Fig. 1. Dendrogram based on 16  analyzed traits in collection Lupinus mutabilis

Table 2. Mean values of morphological and agronomical characters in the groups obtained by cluster analysis
Traits
I
II
III
IV
V
VI
VII
mean
Main stem height [cm]
58.6
67.8
74.2
70.3
68.8
76.1
74.1
70.6
Plant height [cm]
79.3
81.1
85.6
80.8
85.3
90.0
82.7
84.0
Number of lateral branches
3.0
2.7
3.5
3.6
3.8
3.5
2.7
3.3
Inflorescence length of main stem [cm]
19.8
26.0
26.6
24.9
23.9
28.3
29.6
25.7
Whorls number on the main stem
7.7
9.1
8.3
8.3
7.5
9.1
9.6
8.5
Number of  flowers per the main stem
37.8
44.9
42.4
41.5
37.5
45.1
47.0
42.3
Pods number on the main stem
11.4
11.8
12.9
12.7
10.6
11.4
15.4
12.1
Harvest index of the main stem
28.7
25.7
30.4
30.5
28.1
25.8
32.6
28.5
Inflorescence length of the lateral branches [cm]
13.3
13.8
14.0
14.3
12.3
15.8
13.5
13.9
Whorls number on the lateral branches
5.5
5.3
4.7
5.2
4.4
5.6
5.6
5.1
Number of  flowers per lateral stem
27.2
26.0
23.5
26.3
22.0
27.8
26.3
25.4
Number of pods  from branches
7.9
4.8
5.1
5.6
5.2
4.8
7.1
5.5
Harvest index on lateral branches
29.0
18.2
23.0
21.2
23.9
17.9
26.5
22.0
Weight of 1000 seeds main stem [g]
101.1
111.1
169.8
108.0
131.6
140.9
122.0
128.8
Pods number from lateral branches
23.0
13.6
17.8
19.9
19.9
16.7
18.9
18.1
Number of  pods from plant
34.4
25.5
30.6
32.6
30.5
28.1
34.3
30.2
I - Mut-45;  II - 1H_XM.5 x KW, XM.5; III - 5H_LM.13 x KW, 6H_Mut-160 x KW; IV - 2H_XM.5 x KW, 4H_LM.34 x Mut-45; V – LM.34, 3H_LM.34 x Mut-45; VI – LM.13, 7H_MUT-220 x KW; VII – Mut-220

The Andean lupin is not a species easy of adaptation in the edaphic-climatic conditions of Europe. An essential problem, emphasized in earlier papers, was yielding instability resulting from formation by the plants of an exuberant vegetative part [3, 17]. The excessive development of the green mass in the forms of the traditional growth type was responsible for an extension of the period of maturation. And therefore, it was recommendable to undertake research aimed at shortening of the vegetation season, which would involve a change in the plant’s architecture. Like in other cultivated lupin species, after application of mutagenesis, an evenly and early maturing epigonal L. mutabilis mutant ”KW”, characterized by the determined type of growth was obtained [21]. However, the mutant acquired could not be directly used due to its tendency to lodging. The collection assessed in the present paper represents also an outcome of a crossing programme with the use of the above-mentioned epigonal form “KW”, followed by selection of lines which combine the limited growth type with advantageous traits (Table 2, Figure 1).

Simultaneously the results of molecular analysis of SSR and ISSR markers allowed on grouping tested material (Figures 2 A, B, C). The different assignment of the genotypes to groups has been obtained with the application of the two types of DNA markers. The eight tested SSR primers generated 35 polymorphic loci ranging from 184 bp to 300 bp. The 11 tested ISSR primers generated 86 polymorphic products. The number of products amplified using single primers ranged from 4 to 13 with an average of 4.4 products per primer. The size range of the analysed ISSR products was from 250 bp (for primer 824) to 2500 bp (for primer 836). The Nei genetic diversity index equalled 0.91 (Dh) and the Shannon index was 1.0 (I) (Table 3).

Fig. 2
A –
Dendrogram of 12 Lupinus mutabilis genotypes based on ISSR data using the Nei genetic distance matrix and similarity and UPGMA clustering method
B – Dendrogram of 12 Lupinus mutabilis genotypes based on SSR data using the Nei genetic distance matrix and similarity and UPGMA clustering method
C – Dendrogram of 12 Lupinus mutabilis genotypes based on ISSR and SSR data using the Nei genetic distance matrix and similarity and UPGMA clustering method

Table 3. Genetic identity (Is – below the diagram ) and genetic distance (Ds – above the diagonal) statistics for the analyzed genotypes of L. mutabilis
 
XM.5
1H_XM.5 x KW
6H_MUT-160 x KW
Mut-220
7H_Mut-220 x KW
LM.13
5H_LM.13 x KW
LM.34
MUT.45
3H_LM.34 x Mut-45
4H_LM.34 x Mut-45
2H_XM.5 x KW
XM.5
*****
0.446
0.514
0.392
0.522
0.428
0.446
0.121
0.359
0.423
0.538
0.157
1H_XM.5 x KW
0.554
*****
0.496
0.475
0.553
0.51
0.45
0.209
0.324
0.357
0.57
0.141
6H_MUT-160 x KW
0.486
0.504
*****
0.418
0.528
0.445
0.513
0.089
0.338
0.406
0.585
0.172
Mut-220
0.608
0.525
0.582
*****
0.474
0.432
0.423
0.246
0.303
0.36
0.431
0.131
7H_Mut-220 x KW
0.478
0.447
0.472
0.526
*****
0.579
0.463
0.202
0.313
0.428
0.549
0.136
LM.13
0.572
0.49
0.555
0.568
0.421
*****
0.377
0.167
0.357
0.493
0.506
0.164
5H_LM.13 x KW
0.554
0.55
0.487
0.577
0.537
0.623
*****
0.087
0.283
0.318
0.489
0.141
LM.34
0.879
0.791
0.911
0.754
0.798
0.833
0.913
*****
0.18
0.149
0.089
0,000
MUT.45
0.641
0.676
0.662
0.697
0.687
0.643
0.717
0.82
*****
0.348
0.348
0.299
3H_LM.34 x Mut-45
0.577
0.643
0.594
0.64
0.572
0.507
0.682
0.851
0.652
*****
0.393
0.252
4H_LM.34 x Mut-45
0.462
0.43
0.415
0.569
0.451
0.494
0.511
0.911
0.652
0.607
*****
0.145
2H_XM.5 x KW
0.843
0.859
0.828
0.869
0.864
0.836
0.859
1,000
0.701
0.748
0.855
*****

In the case of L. mutabilis, papers concerning usage of molecular markers are relatively scarce. They have mainly been focused on investigation of the phylogenetic relationships within the genus Lupinus [10, 11], and not on the search of markers useful in selectionertaining to given properties. Consequently, the potential of the species as a cultivated plant is not fully made advantage of in the conditions of Europe, where research on its domestication is absolutely worth pursuing. In Peru, for instance, studies have been undertaken to employ SSR and ISSR markers for evaluation of material gathered in the bank of the Andean lupin seeds coming from different locations [4, 5]. Merely 6.67% from the first group of 15 SSR starters gave amplification products, but they were monomorphic and not suitable for estimation of the diversity in the collection. Chirinos-Arias et al. [4] ascertained usefulness of 8 ISSR starters as they generated 255 polymorphic loci. The present paper reveals usefulness for studies on genetic polymorphism not only of ISSR but also of SSR markers. However, application of SSR markers resulted in acquisition of a smaller number of polymorphic loci than in the case of ISSR – 37 and 86 respectively.

The material under analysis clearly displays gradually attained breeding gain [14, 27]. Yet, it still needs some further development, which would provide an opportunity to immediately derive new varieties of the Andean lupin. This requires extension of the pertinent research and introduction of new traits linked with the plants’ immunity to anthracnose and also ones connected with seed quality (protein and fat content, low alkaloid content).

Based on molecular characteristics, a conclusion can be drawn that both types of markers – SSR and ISSR – have revealed wide diversity among the L. mutabilis lines studied. Owing to that, it is possible to choose lines that are the most genetically diverse with respect to DNA polymorphism.

The lack of congruency with morphological markers indicates a need to extend the molecular analysis based on other types of markers, which could prove to be more useful in the process of selection of genotypes that are desirable from the perspective of the species cultivation.

CONCLUSIONS

  1. The first three principal components explain 77.53% of the total multivariate variation of  all the studied traits for 12 evaluated objects.
  2. The first principal component was the most strongly joined with the characteristics connected with construction of the inflorescence and setting pods of evaluated genotypes.
  3. Cluster analysis showed that the evaluated objects are highly different in terms of all 12 evaluated forms. They create several groups of a very high similarity, clearly different from the other Andean lupin populations MUT.45 and MUT.220
  4. Variation in the collection illustrated by the analysis of genetic distances using method of UPGMA does not correspond with differences defined by cluster analysis performed on the basis of the biometric traits analysis.

REFERENCES

  1. Adhikari K.N., Buirchell B.J., Sweetingham M.W. 2012. Length of vernalization period affects flowering time in three lupin species. Plant Breeding 131, 5, 631–636,
  2. Baldeon M.E., Castro J., Villacres E., Narvaez L., Fornasini M. 2012. Hypoglycemic effect of cooked Lupinus mutabilis and its purified alkaloids in subjects with type-2 diabetes.  Nutricion Hospitalaria 27,4, 1261–1266,
  3. Caligari P.D.S., Römer P., Rahim M.A., Huyghe C., Neves-Martins J., Sawicka-Sienkiewicz E.J. 2000. The potential of Lupinus mutabilis as a crop. In R. Knight R. (ed.) Linking Research and Marketing Opportunities for Pulses in the 21st Century. Kluwer Academic Publishers, 569–574
  4. Chirinos-Arias M.C., Jiménez J.E., Vilca-Machaca L.S. 2015. Analysis of genetic variability among thirty accessions of Andean Lupin (Lupinus mutabilis Sweet) using ISSR molecular markers. Scientia Agropecuaria 6, 1, 17-30,
  5. Chirinos-Arias M.C. Jiménez J.E. 2015. Transference of some microsatellite molecular markers from Fabaceae family to Andean Lupin (Lupinus mutabilis Sweet). Scientia Agropecuaria 6, 1, 51–58,
  6. Clements J.C., Sweetingham M.S., Smith L., Francis G., Thomas G., Sipsas S. 2008. Crop Improvement in Lupinus mutabilis for Australian Agriculture – Progress and Prospects. In Palta J.A. and Berger J.B. (eds) Proceding of the 12th International Lupin Conference, 14–18 Sept. 2008, Fremantle, Western Australia, Internationale Lupin Association: 324–327,
  7. Clements J.C., Wilson J., Sweetingham M., Quely J., Francis G. 2012. Male sterility in three crop species. Plant Breeding 131, 155–163,
  8. Clements J.C., Galek R., Kozak B., Michalczyk D.J., Piotrowicz-Cieślak A.I., Sawicka-Sienkiewicz E., Stawiński S. and Zalewski D. 2014. Diversity of selected Lupinus angustifolius L. genotypes at the phenotypic and DNA level with respect to microscopic seed coat structure and thickness. PLoS One 9, 8, 1-8,
  9. Doyle J.J., Doyle J.L. 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13–15,
  10. Eastwood R.J., Hughes C.E. 2008. Origins of domestication of Lupinus mutabilis in the Andes. In Palta JA and Berger JB (eds) Proceding of the 12th International Lupin Conference, 14–18 Sept. 2008, Fremantle, Western Australia, Internationale Lupin Association: 373–379,
  11. Eastwood R.J., Drummond C.S., Schifino-Wittmann M.T., Hughes C.E. 2008. Diversity and evolutionary history of lupins – insights from new phylogenies. In Palta J.A. and Berger J.B. (eds) Proceding of the 12th International Lupin Conference, 14–18 Sept. 2008, Fremantle, Western Australia, Internationale Lupin Association: 346–354,
  12. Falconi C.E., Visser R.G.F., van Heusden S. 2015. Influence of plant growth stage on resistance to anthracnose in Andean lupin (Lupinus mutabilis). Crop and Pasture Science 66, 7, 729-734,
  13. Falconi C.E. 2012. Lupinus mutabilis in Ecuador with special emphasis on anthracnose resistance. PhD thesis, Plant Breeding Laboratory, Wageningen University, The Netherlands, 150p,
  14. Galek R. 2010. Studia nad zmiennością wybranych cech morfologicznych i użytkowych rodzaju Lupinus, ze szczególnym uwzględnieniem mieszańców wewnątrz i międzygatunkowych. [Studies of the variability of some morphological and functional characters of Lupinus with particular consideration intra and intraspecific hybrids]. Dissertation, Wroclaw University of Environmental and Life Sciences, Poland, 122p. [in Polish],
  15. Ghafoor A., Arshad M., 2008. Multivariate analysis for quantitative traits to determine genetic diversity of blackgram Vigna mungo (L.) Hepper germplasm. Pakistan Journal Botany 40(6), 2307–2313,
  16. Legendre P., Legendre L., 2012. Numerical ecology.Elsevier Science BV, Amsterdam, 1006p,
  17. Lema M., Santalla A.P., Rodino A.P., De Ron A.M., 2005. Field performance of natural narrow-leafed lupin from the northwestern Spain. Euphytica 144, 341-351,
  18. Mikić A., Ćupina B., Mihailović V., Krstić D., Antanasović S., Zorić L., Dorević V., 2013. Intercropping white (Lupinus albus) and Andean (Lupinus mutabilis) lupins with other annual cool season legumes for forage production. South African Journal of Botany 89, 296–300,
  19. Nei M. Li W.H., 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases.  In Proceedings of the National Academy Science, USA, 76, 5269–5273.
  20. R Core Team, 2013. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/,
  21. Römer P., Caligari P.D.S., Rahim M.A., Huyghe C., Hardy A., Neves-Martins J., Sawicka-Sienkiewicz E. 1999. Breeding perspectives of Lupinus mutabilis in Middle Europe. In: Hill GD, (ed) Proceedings of the of the 8th International Lupin Conference Towards the 21st Century, 11–16 May 1996; Asilomar, California, USA: North American Lupin Association on behalf of the International Lupin Association, 353–356,
  22. Rybiński W., Rusinek R., Szot B., Bocianowski J. Starzycki M. 2014. Analysis of interspecies physiochemical variation of grain legume seeds. International Agrophysics 28, 491–500
  23. Saini N., Jain N., Jain S., Jain R.K., 2004. Assessment of genetic diversity within and among Basmati and non-Basmati rice varieties using AFLP, ISSR and SSR markers. Euphytica 140(3), 133-146,
  24. Smýkal P., Coyne C.J., Ambrose M.J., Maxted N., Schaefer H., Blair M.W., Berger J., Greene S.L., Nelson M.N., Besharat N., Vymyslický T., Toker C., Saxena R.K., Roorkiwal M., Pandey M.K., Hu J., Li Y.H., Wang L.X., Guo Y., Qiu L.J., Redden R.J., Varshney R.K. 2014. Legume Crops Phylogeny and Genetic Diversity for Science and Breeding. Critical Reviews in Plant Sciences, 34 (1–3), 43–104.
  25. Sweetingham M., Clements J., Buirchell B., Sipsas S., Thomas G., Quealy J., Jones R., Francis C., Smith C.G., 2006. Preliminary breeding and development of Andean lupin for Australian agriculture. In: Santen E and Hill GD (eds) México, where old and new world lupins meet. Proceedings of the 11th International Lupin Conference, Guadalajara, Jalisco, Mexico, 4–9 May 2005, 32–34,
  26. Von Baer E., 2011. Domestication of Andean lupin (L. mutabilis). Lupin crops – an Opportunity for Today, a Promise for the Future. In: Naganowska B., Kachlicki P., Wolko B (eds), Proceedings 13th Internationale Lupin Conference, Poznań, Poland, 129–132.
  27. Zalewski D., Galek R., Kozak B. Sawicka-Sienkiewicz E., 2015. Pheno-morphological and agronomic diversity in a collection of wild and domesticated species of the genus Lupinus. Turkish Journal of Field Crops 20(1), 43–48.

Accepted for print: 30.12.2017


Renata Galek
Department of Genetics, Plant Breeding and Seed Production, Wroc³aw University of Environmental and Life Sciences, Poland
pl. Grunwaldzki 24a
50-363 Wroc³aw
Poland

Bartosz Kozak
Department of Genetics, Plant Breeding and Seed Production, Wroc³aw University of Environmental and Life Sciences, Poland
pl. Grunwaldzki 24a
50–363 Wroc³aw
Poland

Ewa Sawicka-Sienkiewicz
Department of Genetics, Plant Breeding and Seed Production, Wroc³aw University of Environmental and Life Sciences, Poland
pl. Grunwaldzki 24a
50–363 Wroc³aw
Poland
email: ewasawic@ozi.ar.wroc.pl

Dariusz Zalewski
Department of Genetics, Plant Breeding and Seed Production, Wroc³aw University of Environmental and Life Sciences, Poland
pl. Grunwaldzki 24a
50-363 Wroc³aw
Poland

Kamila Nowosad
Department of Genetics, Plant Breeding and Seed Production, Wroc³aw University of Environmental and Life Sciences, Poland
pl. Grunwaldzki 24a
50–363 Wroc³aw
Poland

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