AFLP,RAPD, and ISSR analysis of intraspecific polymorphism and interspecific differences of allotetraploid species <Emphasis Type="Italic">Aegilops kotschyi</Emphasis> Boiss. and <Emphasis Type="Italic">Aegilops variabilis</Emphasis> Eig

To evaluate genetic variation, 27 accessions of allotetraploid species Aegilops kotschyi and Ae. variabilis with the US genome were analyzed using the AFLP, RAPD, and ISSR methods. A total of 316 polymorphic RAPD fragments, 750 polymorphic AFLP fragments, and 234 polymorphic ISSR fragments were obtained. It was demonstrated that the analyzed species were characterized by a considerable level of nuclear genome variation. According to the data of ISSR and RAPD analysis, the average value of the Jaccard similarity coefficient for the accessions of Ae. variabilis from different geographical regions was slightly lower than that for the accessions of Ae. kotschyi. At the same time, AFLP analysis showed no considerable differences in the levels of intraspecific variation of the studied species. Analysis of the summarized RAPD, ISSR, and AFLP marking data in the Structure software program showed that most of the analyzed accessions with high degree of probability could be assigned to one of two groups, the first of which corresponded to Ae. kotschyi and the second corresponded to Ae. variabilis, thereby confirming the species independence of Ae. kotschyi and Ae. variabilis. Accessions k900, k907, k908, and v90 could not with a sufficiently high degree of probability be assigned to one of the species, which possibly was the result of interspecific hybridization. Analysis of the species diversity using different molecular markers made it possible to identify the accessions that were notably different from other accessions of its species.     

Evaluation of genetic relationships in the genus Houttuynia Thunb. in China based on RAPD and ISSR markers

The genetic relationships among 70 accessions of Houttuynia Thunb. from Sichuan, Chongqing, Guizhou and Jiangsu provinces in China were tested using RAPD and ISSR markers. The results showed that the polymorphism of Houttuynia germplasm was high at the DNA level. ISSR markers are more efficient than RAPD markers at uncovering the polymorphism of the genus Houttuynia. The genetic variation between the cultivated and the wild Houttuynia cordata accessions was insignificant according to RAPD and ISSR markers. The results of cluster analysis by using UPGMA method showed that the groups based on ISSR GS was correlated with chromosome numbers and many accessions with the same chromosome numbers could be classified together. Analysis based on RAPD GS was more related to geographic distribution. Furthermore, the cluster analysis based on RAPD and ISSR markers also showed that the genetic diversity in mountainous and margin areas of Sichuan Basin was more plentiful than that at the bottom of the Basin and its surrounding highlands or hills. Houttuynia emeiensis accession could not be separated completely from H. cordata accessions, it was closely related to H. cordata cytotype A with the chromosome number of 36. Within H. cordata, the genetic similarities between each pair of cytotypes C, D, E, F, G, H, I, J, K and L were higher, but the genetic similarities between each of them to the cytotype A were relatively lower. The phylogeny of the germplasm resources of the genus Houttuynia was also discussed.     

Genetic diversity analysis of maintainer and restorer accessions in upland cotton (Gossypium hirsutum L.)

Genetic diversity amongst 91 upland cotton accessions (50 maintainer, ‘B’ and 41 restorer ‘R’ lines) and three wild species viz., G. aridum, G. thurberi and G. anomalum was analyzed using SSR and RAPD markers. A total of 53 primers (30 SSR and 23 RAPD) were sampled for screening 94 accessions, of which 26 SSR and 17 RAPD primers were polymorphic. Average polymorphism detected by SSR, RAPD and SSR + RAPD markers was 72.5, 62 and 66.66 per cent, respectively. A unique marker CIR-200₂₆₀ that distinguishes G. thurberi from all upland accessions has been identified. Similarity coefficient values within and between B and R lines ranged from 0.65–0.95, 0.61–0.98 and 0.53–0.93 for SSR and 0.72–0.98, 0.73–0.97 and 0.69–0.98 for RAPD markers. UPGMA cluster analysis was consistent with the pedigree and genotypic background of the accessions. RAPD and SSR matrices showed significant positive product moment correlation (r?=?0.93 and 0.92) with the RAPD + SSR combined data matrix, respectively. The result indicates a moderate level of genetic diversity in B and R accessions of upland cotton. Genetically diverse combinations were identified to further evaluate heterotic performance. The maintainer, AKH-108, AKH-118 and AKH-2173; and restorer AKH-31 and AKH 4943 accession were identified as most distinct and divergent, could be used as candidate parental genotypes in hybrid and varietal development programme and also development of mapping population for trait mapping in cotton.     

Comparative assessment of variation in the USA Arachis pintoi (Krap. and Greg.) germplasm collection using RAPD profiling and tissue culture regeneration ability

Arachis pintoi accessions were used to study genetic diversity using RAPD markers. Concurrently, two tissue culture protocols were evaluated for organogenesis and the capacity to generate somaclonal variation. Data were collected on callus growth, callus weight gain, and number of regenerated plants. Robust RAPD profiles were obtained and eight primers amplified 100 different bands with 98% polymorphisms. The proportion of polymorphic RAPD loci was 89%. Average genetic distance was 0.36 and indicated that a large amount of genetic diversity exists within the germplasm evaluated. Genetic distances were used to prepare a dendogram for the A. pintoi accessions that separated them into four groups. A large degree of variability for callus induction and callus weight gain was observed among the accessions. Shoot regeneration was achieved for several accessions on both media with no structures indicative of somatic embryogenesis detected. Root induction was difficult to obtain, and many shoots died during this process. RAPD band profiles of regenerated tissue culture plants were similar to their parent plants, and therefore no somaclonal variation was evident using these methods.     

Assessment of genetic diversity in Colletotrichum falcatum Went accessions based on RAPD and ISSR markers

Sugarcane is susceptible to red rot disease caused by phytopathogenic fungus Colletotrichum falcatum Went which ultimately affect the economy of farmers as well as sugar based industry. One of the various ways to control this devastating disease is to develop disease resistance sugarcane cultivar and this requires the complete understanding of genetic makeup of pathogen. Although South Gujarat is well known sugarcane cultivating area, less published data can be found about PCR-based genetic diversity in prevalent C. falcatum accessions. So, present investigation aims at finding molecular variation among the ten accessions of C. falcatum using RAPD and ISSR molecular markers. A total of 35 RAPD and 39 ISSR primers were screened across 10 C. falcatum accessions, of which 15 RAPD and 21 ISSR primers have showed consistent amplification. Statistics related to genetic variation were estimated using NTSYS-PC by means of Dice’s coefficient. The results revealed 80.6% and 68.07% polymorphism and similarity coefficient ranged from 0.43 to 0.91 and 0.73 to 0.93 in RPAD and ISSR analysis respectively. The dendrogram generated using RAPD, ISSR and combined RAPD-ISSR grouped accessions into different clusters which reveal considerable level molecular variation among the C. falcatum accessions. It is also evident from PCA plots that accessions are rather dispersed with tested marker systems indicating good genetic base. So, in nut shell, we found considerable genetic variation and relatedness within C. falcatum accessions collected from different areas of south Gujarat, India using RAPD and ISSR markers.     

Genetic Relationships Among Wild and Cultivated Accessions of Curry Leaf Plant (Murraya koenigii (L.) Spreng.), as Revealed by DNA Fingerprinting Methods

Murraya koenigii (L.) Spreng. (Rutaceae), is an aromatic plant and much valued for its flavor, nutritive and medicinal properties. In this study, three DNA fingerprinting methods viz., random amplification of polymorphic DNA (RAPD), directed amplification of minisatellite DNA (DAMD), and inter-simple sequence repeat (ISSR), were used to unravel the genetic variability and relationships across 92 wild and cultivated M. koenigii accessions. A total of 310, 102, and 184, DNA fragments were amplified using 20 RAPD, 5 DAMD, and 13 ISSR primers, revealing 95.80, 96.07, and 96.73% polymorphism, respectively, across all accessions. The average polymorphic information content value obtained with RAPD, DAMD, and ISSR markers was 0.244, 0.250, and 0.281, respectively. The UPGMA tree, based on Jaccard’s similarity coefficient generated from the cumulative (RAPD, DAMD, and ISSR) band data showed two distinct clusters, clearly separating wild and cultivated accessions in the dendrogram. Percentage polymorphism, gene diversity (H), and Shannon information index (I) estimates were higher in cultivated accessions compared to wild accessions. The overall high level of polymorphism and varied range of genetic distances revealed a wide genetic base in M. koenigii accessions. The study suggests that RAPD, DAMD, and ISSR markers are highly useful to unravel the genetic variability in wild and cultivated accessions of M. koenigii.     

Diversity among wild accessions of Bacopa monnieri (L.) Wettst. and their morphogenetic potential

Biochemical and molecular diversity among 14 accessions of Bacopa monnieri (L.) Wettst. collected from various locations of India was investigated. A significant variation was recorded in bacoside A contents of these accessions. A scatter plot of principle component analysis based on bacoside A contents clubbed these populations into two major groups and accession BM14 was placed separately. Similarly, about 35 % variations were detected in these populations based on combined data of random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR). Individually, ISSR markers detected higher variation (44.9 %) as compared to RAPD markers (23 %). Clustering based on molecular marker data grouped these accessions into two major groups and also placed accession BM14 as an out group. The shoot organogenic potential of leaf explants taken from microshoots and rooting of microshoots also varied among accessions. Maximum shoot organogenic potential was observed in accession BM5 and maximum rooting potential was observed in accessions BM1, BM2, BM7, BM10 and BM14. Present study is an important step for developing long-term strategy for conservation of this important medicinal herb.     

Assessment of genetic diversity and morpho-physiological traits related to drought tolerance in Stylosanthes scabra

Among the many Stylosanthes species, Stylosanthes scabra, a range fodder legume, performs better under limited water condition. In the present investigation, thirty-four accessions of S. scabra were assessed under limited water condition, for various morpho-physiological characters associated with drought. In general, S. scabra exhibited better tolerance to drought, as evidenced by high leaf thickness and greater accumulation of proline, and malondialdehyde (MDA) in water stress condition. Transpiration efficiency (TE) was high, in both control and water stress conditions and positively correlated with root, shoot, and total dry matters, in both control and stress conditions (r ² = ranged from 0.589 to 0.961 in control and from 0.351 to 0.985 in stress). Of these, 25 accessions were assessed for estimation of genetic diversity, employing random amplified polymorphic DNA (RAPD) markers. A total of 210 RAPD bands, obtained with 32 primers, revealed high polymorphic information content (0.49) and marker index (4.41). Dendrogram analysis indicated close proximity among the accessions of S. scabra. These accessions were clustered in high similarity range (84.01–98.36 %). Accession IG-366A separated from other clusters at 85.62 % similarity level. RAPD marker system revealed 13 accessions exhibiting >90 % genetic similarity while the other accessions exhibited similarity ranging from 68 to 90 %. A higher level of genetic similarity which was also evident from the similar levels of TE, biomass production, root/shoot ratio, MDA, proline contents and drought tolerance index, indicated a cause–effect relationship among them. Results also indicated that among the accessions, S. scabra rate-reducing resistance allo-tetraploid lines were better suited for hard and cracking soils, under complete rain-fed condition.     

Analyses of genetic relationships in Nelumbo nucifera using nuclear ribosomal ITS sequence data,ISSR and RAPD markers

Despite the economic importance of Nelumbo nucifera, there have been no molecular studies on genetic relationships among cultivars in the species. In the present study 38 accessions were sampled including 37 accessions of N. nucifera or hybrids between N. nucifera and Nelumbo lutea and a single accession of N. lutea. In the ITS analyses, Chinese and Japanese lotus comprise a single cluster with a moderate bootstrap support 68% indicating there is very high similarity between them. Moreover, these ISSR and RAPD results also indicate that there is very close genetic relationship between Chinese and Japanese lotus. In the ISSR and RAPD analyses, although 38 accessions all are distinctly separately into two groups, viz. N. nucifera and N. lutea, there is a high Jaccard similarity coefficient (0.785 and 0.656) between the two species. In N. nucifera the two different groups of the species, viz. flower lotus and rhizome lotus accessions show clear genetic variations. Seed lotus accessions do not form a distinct cluster but are interspersed among the flower accessions indicating that seed lotus is phylogenetically close to flower lotus and they might originate from close wild lotus in genetic relationship. In flower lotus, big-flower type accessions and medium-small type accessions have obvious genetic variation, indicating height is an important criterion in the classification system of flower lotus.     

Analysis of molecular genetic diversity in a representative collection of foxtail millet [Setaria italica (L.) P. Beauv.] from different agro-ecological regions of India

Foxtail millet [Setaria italica (L.) P. Beauv.], an important crop of East Asia is known for its drought tolerance and was once an indispensible crop of vast rainfed areas in semi-arid regions in India. In India it is cultivated in Andhra Pradesh, Karnataka, Maharashtra, Tamil Nadu, Rajasthan, Madhya Pradesh, Uttar Pradesh and north eastern states. The grain finds use in several local recipes such as roti (bread), jaula, singal, sirol. Foxtail millet grain contains 12.3 % protein, 4.7 % fat, 60.6 % carbohydrates, and 3.2 % ash. The present study was conducted to analyse the genetic diversity among foxtail accessions from different states of India and a few exotic accessions using RAPD and ISSR techniques and identify diverse accessions for use in variety improvement programmes. A set of 125 foxtail millet accessions selected from 11 different agro-ecological regions of India were analyzed using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) marker techniques. A total of 146 (115 RAPD and 31 ISSR) scoreable markers were generated with 16 RAPD and four ISSR primers. The dendrogram generated using Nei’s genetic distances and principal component analyses revealed presence of two clusters and two subclusters in group I. The accessions from Andhra Pradesh, Karnataka, Maharashtra and Uttarakhand were more diverse since they were distributed in both the clusters. There was no clear geographical differentiation observable. The bootstrap support for the major groups identified was strong (above 80 %) indicating good statistical support. The average value of Nei and Li’s genetic distance was lowest (0.081) for accessions from West Bengal while the collections from Karnataka showed highest dissimilarity (average genetic distance = 0.239). The average genetic distance for all 125 accessions together was 0.177 indicating presence of only moderate genetic diversity in the collections. The analysis of molecular variance indicated that only 2.76 % variation was explained by variations among the groups and 11.55 % among populations within groups. However the percentage of variation observed within populations was high (85.68). The value of Fst was observed to be very low (0.028) indicating low differentiation of the accessions analysed. The population genetic analysis carried out indicates that highest number of alleles per locus (1.745 ± 0.438) was observed for Andhra Pradesh with 35 accessions. When four eco-geographic regions were considered, the southern region comprising AP, Karnataka and TN showed the highest number of alleles per locus (1.787 ± 0.411). The value of Gst was lowest for south (0.123) and highest for central west (0.455). This indicated that all the landraces from south share common alleles. The gene flow between the accessions from different regions was also observed to be high with the highest migration (3.557) recorded for south.     

Genetic diversity and genetic relationships in Hyacinthaceae in India using RAPD and SRAP markers

Genetic diversity and relationship among three genera namely Drimia, Dipcadi and Ledebouria of Hyacinthaceae in India was studied using RAPD and SRAP markers. Twenty one RAPD primers and nine SRAP were used for analyzing 41 accessions. RAPD gave an average 12.6 markers per primer, while SRAP generated 10.1 markers per primer pair. The family emerged very diverged with high polymorphism. The study resolved the three genera into monophyletic groups corresponding to three subfamilies; Urginoideae, Hyacinthoideae and Ornithogaloideae. Drimia wightii emerged a very distinct species and species specific markers were obtained with both marker systems. AMOVA analysis also revealed the genera to be quite well diverged. The two markers showed high correlation (r = 0.932) in Mantel matrix crresspondance test. The combined data also showed a very good correlation with the respective markers individually.     

Molecular genetic marker-based approaches to the verification of lilac Syringa vulgaris L. in vitro germplasm collections

RAPD analysis was used to verify the varieties in an in vitro germplasm collection of lilac Syringa vulgaris L. RAPD patterns were obtained with 16 decanucleotide primers for 46 accessions (microclones and corresponding reference varieties). The RAPD patterns of a microclone and the corresponding reference variety often differed in composition; consequently, it was infeasible to verify the accessions by direct comparison of the RAPD patterns. Hence, evaluation of the relative genetic distances between accessions (microclones) and known varieties was proposed as a method to verify lilac in vitro germplasm collections.     

Analysis of variations in RAPD profiles among accessions ofProsopis

The genetic variability among accessions ofProsopis was determined using randomly amplified polymorphic DNA (RAPD) profiles. Similarities of profiles were determined using the algorithm of Jaccard, and UPGMA and neighbour joining trees were generated from the similarity data. The average similarity was highest among the accessions ofP. glandulosa (0.52 ± 0.18) and least in the accessions ofP. juliflora (0.37 ± 0.15), indicating that the latter species has greater diversity among accessions. Our observations suggest that RAPD analysis could help in identifying genetic variations among different accessions ofProsopis.     

Analysis of Genetic Diversity of Persea bombycina “Som” Using RAPD-Based Molecular Markers

The utility of RAPD markers in assessing genetic diversity and phenetic relationships in Persea bombycina, a major tree species for golden silk (muga) production, was investigated using 48 genotypes from northeast India. Thirteen RAPD primer combinations generated 93 bands. On average, seven RAPD fragments were amplified per reaction. In a UPGMA phenetic dendrogram based on Jaccard’s coefficient, the P. bombycina accessions showed a high level of genetic variation, as indicated by genetic similarity. The grouping in the phenogram was highly consistent, as indicated by high values of cophenetic correlation and high bootstrap values at the key nodes. The accessions were scattered on a plot derived from principal correspondence analysis. The study concluded that the high level of genetic diversity in the P. bombycina accessions may be attributed to the species’ outcrossing nature. This study may be useful in identifying diverse genetic stocks of P. bombycina, which may then be conserved on a priority basis.     

Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.     

Genetic Relationships Within and Between Capsicum Species

Genetic relationships were estimated among 24 accessions belonging to 11 species of Capsicum, using 2,760 RAPD markers based on touch-down polymerase chain reactions (Td-RAPD-PCR). These markers were implemented in analyses of principal coordinates, unweighted pair group mean average, and 2,000 bootstrap replications. The accessions were divided into four groups, corresponding to previously described Capsicum complexes: C. annuum complex (C_A), C. baccatum complex (C_B), C. pubescens complex (C_P), and C. chacoense accessions (C_A/_B). Their overall mean genetic similarity index was 0.487 ± 0.082, ranging from 0.88 to 0.32, based on Jaccard’s coefficient. The highest genetic variation was observed among the accessions in C_P; the accessions in C_B had a low level of variation as judged from the standard deviations of the genetic similarity indices. Based on the Td-RAPD-PCR markers, the 24 accessions were divided into four major groups, three of which corresponded to the three distinct Capsicum complexes. Accessions of C. chacoense were found to be equally related to complexes C_A, C_B, and C_P.     

Assessment of Genetic Diversity and Identification of Informative Molecular Markers for Germplasm Characterization in Caribbean Stylo (Stylosanthes hamata)

Caribbean stylo (Stylosanthes hamata) is a tropical fodder and cover crop. Along with four other Stylosanthes species (S. scabra, S. humilis, S. viscosa, S. guianensis), it was introduced in India. It became well adapted in certain parts of the country and has been recommended for the improvement of range and degraded lands. A collection of 63 S. hamata accessions was fingerprinted with RAPID, ISSR and STS markers. Though the mean discriminating power of these marker systems ranges from 0.65 to 0.71, high values of marker index (2.91), resolving power (14.92) and effective number of patterns per assay unit (50.65) makes ISSR as a better marker system in comparison to other two markers used in this study. Thirteen RAPD and eleven STS primers could differentiate a maximum of 42 and 17 accessions, respectively, whereas two ISSR primers produced distinct fingerprints of all the S. hamata accessions. Mean genetic similarities of accession ranged from 0.83 (ISSR) to 0.91 (RAPD). Two RAPD, two STS and four ISSR primers generated a set of 12 diagnostic markers which could be useful for germplasm characterization and management.     

Inter and intraspecific genetic diversity (RAPD) among three most frequent species of macrofungi (Ganoderma lucidum,Leucoagricus sp. and Lentinus sp.) of Tropical forest of Central India

In present study seven RAPD primers were used to access the diversity within and among twelve populations of three mushroom species Ganoderma lucidum, leucoagaricus sp. and Lentinus sp. Total of 111 bands were scored by 7 RAPD primers in 30 accessions of three mushroom species collected from different sampling sites of central India. Total 111 bands were generated using seven primers which were F-1, OPG-06, OPC-07, OPD-08, OPA-02, OPD-02, OPB-10. All 111 bands were polymorphic in nature (100%). Therefore, it revealed that the used primers had sufficient potency for population studies and 30 accessions had higher genetic differences among each other. In best of the knowledge, this is the first report, which accesses the genetic diversity between three mushroom species (Gd Ganoderma lucidum, Lg Leucoagaricus sp., Ls Lentinus). The polymorphic percentage ranged from 3.60 to 23% within twelve populations, while polymorphic percentage among group was 40.56, among population within groups was 41.12 and within population was 18.32. This indicated that the genetic diversity within the population was very low, but slightly higher in the populations of three species. Among three groups representing Gd., Lg and Ls, Among populations within groups shown highest percentage of variation (Pv?=?41.12) while within populations, the lowest percentage of variation (18.32) was observed. This result also support that the highest genetic variation was present among groups in comparison to among the population within a species and lowest genetic variation was observed within the population.     

Genetic Diversity of Radish (Raphanus sativus L.) Germplasm Resources Revealed by AFLP and RAPD Markers

Genetic diversity of 56 radish accessions, representing nearly all the typical types and origins of cultivated radish germplasms conserved in the National Mid-term Genebank for Vegetables of China, was assessed with amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) markers. A total of 72 and 128 polymorphic bands were generated by the 12 selected RAPD primers and eight AFLP primer combinations respectively. A moderate correlation with the value of r = 0.66 was observed between AFLP and RAPD markers. The total 200 polymorphic bands were integrated to assess the genetic diversity of 56 radish accessions. The Jaccard similarity coefficients between the accessions varied from 0.30 to 0.83 with the mean of 0.54. Cluster analysis classified the germplasms into three groups of var. hortensis Becker, var. sativus, and var. niger Kerner. The three-dimensions scatter plot of principle coordinate analysis (PCA) further divided var. hortensis Becker germplasms into two separate groups. The results indicated that the genetic diversity harbored among var. hortensis Becker germplasms was very abundant, which could be further exploited for radish genetic improvement.     

Identification and relationships of cultivated accessions fromLolium-Festuca complex based on RAPD fingerprinting

Randomly Amplified Polymorphic DNA (RAPD) technique with 15 arbitrary primers was used to identify and reveal relationships of the accessions comprising 4 species ofLolium-Festuca complex. Altogether 252 RAPD markers were considered in statistical treatment, 60 of which could be identified as potentially taxon-specific. All analyzed taxa were fully distinguishable using RAPD markers.Lolium-Festuca relationships based on RAPD data were evaluated using cluster analysis (UPGMA) and principle coordinate analysis (PCO). The results of UPGMA as well as PCO performed on data pooled from all RAPD profiles support separation of the two generaLolium andFestuca.