Genetic Diversity of the Vigna Germplasm from Thailand and Neighboring Regions Revealed by AFLP Analysis

Thailand is a center of diversity for section Angulares of the Asian Vigna (genus Vigna subgenus Ceratotropis) and 4 Vigna species are cultivated in Thailand. Using newly collected wild and cultivated germplasm of Vigna from Thailand and outgroup accessions AFLP analysis was conducted to clarify genetic diversity and relationships. The results suggest that cultivated V. umbellata and V. mungo evolved from wild relatives in a single domestication event. Vigna umbellata is poorly differentiated from its wild and weedy relatives compared to V. mungo. Results suggest northern Thailand and the neighboring Shan state, Myanmar, is the probable center of domestication for V. umbellata as wild accessions from this area and cultivated rice bean from a wide area in Asia are not greatly diverged. Vigna minima, V. tenuicaulis and V. exilis accessions in Thailand are well differentiated with considerable intra-specific variation. Vigna hirtella consists of two well differentiated subgroups, suggesting taxonomic revision may be necessary. Close genetic relationships between V. radiata and V. grandiflora, and between V. mungo and V. trinervia are confirmed. Naturally growing V. mungo populations in northern Thailand appear to be true wild species as they are well differentiated from Indian wild and Thai cultivated populations. The origin of naturally growing cowpea in Thailand needs to be further studied using a more comprehensive set of materials. This study clarifies inter and intra-specific genetic diversity and inter species relationships of Thai Vigna species.     

Genetic Diversity among Syrian Cultivated and Landraces Wheat Revealed by AFLP Markers

Genetic diversity among some important Syrian wheat cultivars was estimated using Amplified Fragment Length Polymorphism (AFLP) markers. Five Triticum aestivum L. and 10 Triticum turgidum ssp. durum were analyzed with 11 EcoRI–MseI primer pair combinations. Of the approximately 525 detected AFLP markers, only 46.67% were polymorphic. Cluster analysis with the entire AFLP data divided all cultivars into two major groups reflecting their origins. The first one contained T. aestivum L. cultivars, and the T. turgidum ssp. durum cultivars and landraces were grouped in the second. Narrow genetic diversity among all cultivars was detected with an average genetic similarity of 0.884. The lowest similarity index (0.9) was found between Cham5 and Hamary (durum wheat), whereas this value was 0.93 between Salamony and Bouhouth 4 (T. aestivum L.). The narrow genetic diversity level indicates that these genotypes could be originated from the same source. AFLP analysis provides crucial information for studying genetic variation among wheat cultivars and provides important information for plant improvement.     

AFLP Analysis of the Phenetic Organization and Genetic Diversity in the Sugarcane Complex, Saccharum and Erianthus

Amplified fragment length polymorphism (AFLP) markers were evaluated for determining the phylogenetic relationships, and the diversity in the Saccharum complex using 30 clones belonging to S. officinarum, S. robustum, S. spontaneum, S. barberi, S. sinense and the related genus Erianthus. The phenetic tree of the species clones based on AFLP data was consistent with the known taxonomical relationships. AFLP gave higher resolution of closely related species into discrete groups than that by RAPD and RFLP markers, reported earlier. The levels of diversity within the various Saccharum species were also found to be higher than those obtained previously with the same set of clones using RAPD markers. The intraspecies similarity in S. barberi and S. sinense was much higher than interspecies similarity suggesting a clear separation of the two, which are considered ‘horticultural species’. The genetic similarity matrix derived from a single primer combination highly correlated (r = 0.980) with that obtained from all the 12 primer combination used in the study, thus highlighting the efficiency of a single primer combination in delineating species relationships. All the primer combinations could identify markers that are specific to each of the species and the genus Erianthus. Among the species, specific markers were highest in S. spontaneum followed by S. robustum, S. barberi, S. officinarum and S. sinense. Erianthus had a distinct profile with 30% of the total amplified fragments being specific to it. This offers great scope for identifying intergeneric hybrids, which has been very difficult using morphological traits and RAPD markers. High degree of correspondence between the results from the cluster analysis based on Jaccard's similarity index, Neighbour Joining tree based on Sokal and Michener distance matrix and AFTD (Analyses Factorielle on Table of Distances) analysis clearly demonstrated that AFLP markers would be an appropriate tool in providing better information about the relationships among the species, estimation of diversity, and in revealing species and genus specific markers that could be directly applied in sugarcane breeding programmes.     

AFLP Analysis of Genetic Diversity in Indian Soybean [Glycine max (L.) Merr.] Varieties

AFLP technique was used to assess genetic diversity in 72 soybean varieties under cultivation in India. Selected 12 AFLP primer pairs produced 1319 products of which 1257 were polymorphic (95%). Wide variations were observed for the number of amplification products, percent polymorphism and average polymorphism information content (PIC). The Jaccard's similarity indices (J) based on the AFLP profiles of the 72 soybean varieties were subjected to UPGMA cluster analysis. The dendrogram generated revealed four major clusters, which were strongly supported by the high bootstrap values obtained from analyses of 1000 bootstrap samples. In addition, the Mantel's test for cophenetic correlation with r = 0.955 indicated very good fit of the varieties to a group in the cluster analysis. Some correspondence between the clustering pattern and the pedigree, place of release or target area of the variety was observed. Overall moderately high genetic diversity was observed which appears to be due to the higher genetic diversity prevalent in 12 of the varieties included in three diverse clusters and was indicative of the need to include more diverse germplasm in the soybean improvement programs.     

Microsatellite Markers Revealed the Genetic Diversity of an Old Japanese Rice Landrace ‘Echizen’

Landraces of rice (Oryza sativa L.) are valuable sources of genetic variation that have been lost in advanced cultivars. Seeds of a rice landrace stored for almost 100 years were found on Sado Island in Niigata prefecture, Japan. This report aims to present basic data on the genetic variation of this landrace, which was known as ‘Echizen’. Five samples of ‘Echizen’, consisting of two old samples, one sample maintained on farm, and two lines regenerated from old seeds were compared with other advanced cultivars and landraces using 19 microsatellite markers. Among the five samples of Echizen, the two stored samples showed greater diversity than the other samples. Cluster analysis based on the UPGMA method also showed that old Echizen was a diverse landrace that could cover the genetic diversity of most Japanese rice cultivars.     

四个鲤鱼种群遗传多样性的AFLP分析

本文采用AFLP技术对黑龙江野鲤、黄河鲤、建鲤和荷包红鲤4个鲤鱼种群共96个个体进行了遗传多样性分析。结果显示,8对选择性扩增引物共扩增得到502个位点,其中多态性位点273个,多态性比率为54．38％。同时对4个种群的Shannon多样性指数,Nei’s基因多样性等参数进行了分析,结果表明,4个种群的Shannon多样性指数分别为0．2114±0．2705,0．1825±0．2694,0．1888±0．2587和0．1600±0．2426,Nei’s基因多样性指数分别为0．1398±0．1872,0．1225±0．1863,0．1235±0．1774和0．1036±0．1636;总基因多样性（H4）平均值为0．1721±0．0350;种群内基因多样性（Hs）平均值为0．1224±0．0190;基因分化系数（Gst）为0．2892,种群内的基因多样性占总群体的71．08％,种群间为28．92％,而基因流系数（Nm）为1．2291。另一方面,分子方差分析（AMOVA）结果表明,种群平均近交系数（Fst）为0-31191,变异31．19％来自种群间,68．81％来自种群内。4个种群中黑龙江野鲤的种内多态性比例最高,而荷包红鲤种群最低,并且4个鲤鱼种群当前的种质资源良好,具有一定的种群稳定性;建鲤已经开始分化,与亲本荷包红鲤亲缘关系逐渐分化,逐步形成自己稳定的遗传结构。本研究为探讨鲤鱼种群的遗传特性和遗传分化提供参考,也为其种质资源的保护及合理利用提供科学依据。     

Genetic Diversity within and Between Populations of Lathyrus genus (Fabaceae) Revealed by ISSR Markers

In order to evaluate the genetic diversity in Lathyrus genus, the Inter Simple Sequence Repeats method (ISSR) was exploited in five populations. These consisted of two cultivated species belonging to section Lathyrus (L. sativus L. and L. cicera L.) and a wild one belonging to the section Clymenum (L. ochrus DC.). Two 3′anchored ISSR primers and two unanchored ones, generated a total of 60 useful polymorphic DNA bands. Our data provide evidence of high molecular polymorphism at the intra- and the inter-specific levels showing that both wild and cultivated forms constitute an important pool of diversity. Moreover, among the generated DNA bands, a 500 bp band, totally absent in the banding patterns of the section Clymenum, appears to be a molecular marker of section Lathyrus. Results provided for lineage and suggest recent origin of these species that might have evolved from a common ancestor producing both L. ochrus species and the two other species L. sativus and L. cicera. These relationships support previous studies based on morphological variation and molecular analysis.     

紫茎泽兰群体遗传多样性及遗传结构的AFLP分析

紫茎泽兰是我国最为严重的入侵物种之一。本研究以中国境内的紫茎泽兰为材料，用AFLP方法研究了其群体遗传多样性及遗传分化。用筛选出的3对荧光引物，对5个地区62个群体进行AFLP分析，共扩增出490条带，其中多态性带328条，群体的遗传多样性较高(P = 66.9%，H = 0.171，I = 0.268)，主要存在于群体内，群体间的遗传分化系数Fst为0.287。AMOVA分析表明，在地区水平紫茎泽兰的遗传分化主要存地区内，群体内遗传分化占 70.25%，群体间遗传分化占21.71%，地区间的遗传分化仅占8.04%。UPGMA聚类分析结果表明，62个地理群体主要分为四大类群，并且有明显的地缘关系。Sigmaplot分析表明海拔是影响紫茎泽兰遗传多样性主要地理因子。Mantel检验表明遗传距离与地理距离成正相关(r = 0.34)，但也有例外。由此推断风媒传播可能是紫茎泽兰的主要传播方式，水媒传播可能是紫茎泽兰的另一主要传播途径；随着紫茎泽向东、向北扩散，新入侵地区的遗传多样性逐步降低。     

Genetic Diversity and Relationships of Japanese Peach (<Emphasis Type="Italic">Prunus persica</Emphasis> L.) Cultivars Revealed by AFLP and Pedigree Tracing

To evaluate the genetic diversity and to clarify the genetic relationships of Japanese peach cultivars, we analyzed the amplified fragment length polymorphism (AFLP) and traced the pedigree of 17 Japanese commercial peach cultivars and six traditional accessions. Sixteen AFLP primer combinations produced a total of 837 fragments and 146 polymorphic bands with a polymorphism percentage of 17.5%. All of the peach accessions could be identified from differences in at least 10 polymorphic bands. A cluster analysis showed that all the Japanese commercial peach cultivars, except ‘Kiyomi’ and ‘Jichigetsuto’, formed a major group consisting of three sub-groups. Of the six traditional accessions, four were genetically distant from the Japanese commercial peach cultivars while two accessions from China were classified into the Japanese commercial peach cultivars group. Both the AFLP analysis and pedigree tracing suggested that Japanese commercial peach cultivars are mainly derived from ‘Shanhai Suimitsuto’, one of the traditional accessions from China. Although the genetic relationships revealed by AFLP were generally in agreement with those shown by the pedigree information, some contradictions were found. Combining the AFLP results and pedigree information can provide a better understanding of the genetic relationships of Japanese peach cultivars.     

Analysis of Genetic Diversity in Colonial Bentgrass (<Emphasis Type="Italic">Agrostis capillaris</Emphasis> L.) using Randomly Amplified Polymorphic DNA (RAPD) Markers

Colonial bentgrass (Agrostis capillaris L.) is a cool-season grass, native to temperate Asia and Europe. It has good tolerance to low temperatures and partial shade and is well suited to golf course fairways and tees. Little information is available regarding levels and patterns of genetic variation among populations of colonial bentgrass, which would be useful for breeding programs. To study the genetic relationships among 27 colonial bentgrass accessions obtained from the US National Plant Germplasm System (NPGS), randomly amplified polymorphic DNA (RAPD) markers were scored and analyzed. Out of 80 primers screened, 16 were selected for further analysis, which yielded a total of 120 polymorphic bands used to differentiate the accessions. Dice's similarity coefficients for pair-wise comparisons ranged from 0.23 to 0.84 based on the RAPD data. Since there was no similarity coefficient value close to 1 between any two accessions, there was no apparent duplication among the sampled accessions. A dendrogram constructed on the basis of the Unweighted Pair Group Method with Arithmetic average (UPGMA) clustering algorithm clearly separated 26 of the accessions into three clusters with one accession distinct from the rest. The least similar pair of accessions was PI 204397 from Turkey and PI 628720 from Bulgaria, and the most similar pair was PI 509437 from Romania and PI 491264 from Finland. Clustering patterns based on principal components analysis (PCA) corresponded well with the dendrogram. A high cophenetic correlation (r = 0.82) was found between the RAPD data matrix and cophenetic matrix. The accession PI 628720, from Bulgaria, did not cluster with any other accessions.     

DNA Fingerprinting and Genetic Characterization of Anatolian Triticum spp. using AFLP Markers

In this study, genetic analysis of Triticum spp. was carried out using AFLP markers. Six AFLP selective combinations were scored as presence and absence of bands for all the individual samples obtained from a single seed of each accession (70 accessions); T. baeoticum (21), T. monococcum (5), T. urartu (16), T. araraticum (7), T. dicoccoides (16) and T. dicoccon (5), resulting in 506 polymorphic AFLP bands. The phylogenetic tree showed two major clusters; one was composed of T. monococcum (AA) and T. baeoticum (AA), and the other cluster included T. araraticum (AAGG), T. dicoccon (AABB), T. dicoccoides (AABB), and T. urartu (AA). T. urartu, although having a diploid AA genome, did not cluster with other A genome diploids such as T. monococcum and T. baeoticum; instead it clustered together with the tetraploid species, confirming that T. urartu is the A genome progenitor. The extent of variations within and among species is discussed.     

Genetic Diversity Among Brazilian Cultivars and Landraces of Tomato Lycopersicon Esculentum Mill. Revealed by RAPD Markers

Random amplified polymorphic DNA markers (RAPD) were used to estimate the variability of 35 tomato accessions (Lycopersicon esculentum Mill.). A total of 257 reproducibly scorable bands were obtained from 20 primers, 78.6% of which were polymorphic. The percentage distribution of RAPD markers shows a bimodal distribution, and the frequency of rare alleles is similar in commercial and landrace accessions. Genetic distances among accessions were calculated and a dendrogram showing the genetic relationships among them was constructed allowing for the separation of four groups. Twenty out of 23 Brazilian landraces fell within one group, whereas commercial cultivars were distributed in the four groups. AMOVA analysis of RAPD data showed that, despite the high within Brazilian landraces and commercial cultivars variation, these two groups are significantly different, indicating that landraces can be a source of variation for breeding programs.     

我国甘蔗亲本遗传多样性的AFLP标记分析

采用15对多态性较好的AFLP引物对我国自育的78个甘蔗亲本材料遗传多样性进行分析,结果表明,每对引物的多态性位点平均为65．67,多态陆位点率为82．55％。78个甘蔗亲本的遗传相似系数在0．4535～0．8927之间,平均为0．6821。相同组合后代的遗传相似系数较高,平均为0．7656。以遗传相似系数阈值约为0．700划分,聚类分析把78个亲本分为7大类,系谱记录中亲缘关系密切的亲本品系,大多数都能归为同一类。遗传多样性指数分析显示,不同年代亲本多样性指数差异明显,20世纪80年代最高,为0-3185,70年代最低,为0．2645;不同省区的亲本遗传多样指数变化更显著,在0．1952～0．2999之间,广东最高,云南最低。     

马氏珠母贝4个壳色选育系F1遗传结构的AFLP分析

本研究从湛江流沙港马氏珠母贝养殖群体中挑选不同壳色个体为亲本,共构建了黑（BS）、红（RS）、黄（YS）和白壳色（WS）4个壳色选系F1,然后分别在4个壳色选育系F,中随机取样,利用3对AFLP引物分析其遗传结构与遗传分化。结果表明,每对引物的扩增位点数在104～109之间,共得到331个扩增位点;BS、RS、YS和WS壳色选系F1的多态位点比例分别为49．2％、49．5％、51．6％和63．4％,Shannon’s多样性指数分别为0．1884、0．1886、0．1896和0．1954,4个壳色选育系F1的遗传分化系数（Fst）为0．1972。本研究说明经过一代壳色纯化4个壳色选育系F1出现显著遗传分化,也为马氏珠母贝壳色系选育或选育系间杂交提供了参考依据。     

Genetic Diversity of Traditional Chinese Mustard Crops Brassica juncea as Revealed by Phenotypic differences and RAPD Markers

This investigation was aimed at exploring the genetic diversity among nine typical accessions of Chinese mustard crops using random amplified polymorphic DNA (RAPD) markers and morphological comparison. Totally, 111 reproducible DNA bands were generated by 16 arbitrary primers, of which 91 bands were proved to be polymorphic. Based on pair-wise comparisons of the amplified bands, genetic similarities were obtained using Nei & Li's similarity coefficients and a dendrogram reflecting their relationships was made using the unweighted pair–group method with arithmetic averages (UPGMA). The result of cluster analysis indicated that the nine accessions were capable of being classified into two primary groups, one including accession 2 with expanded root (root mustard), accession 3 with entirely expanded whole stem (long-stem mustard), accession 6 with edible leaves (leaf mustard), accession 8 with edible seed stalk (seed stalk mustard) and another one including accession 4 with expanded basal stem (short-stem mustard), accession 5 with bulgy petiole (leafy bulgy mustard), and accession 9 with mustard-rich seed (seed mustard). Besides, accession 1 with expanded root (root mustard) and accession 7 with edible leaves and seed stalk (seed stalk mustard) were independent of other accessions in the dendrogram. Additionally, by cluster analysis based on highly reproducible RAPD markers, the accessions with similar edible parts of leaves or roots were not actually in the same phylogenetic groups. This implied that they were probably derived from different geographical origins with dissimilar genetic background and possessed higher genetic diversification. Furthermore, the results indicated that the traditional method for classifying Chinese mustard crops was not much reliable as it was largely dependent on phenotypic behaviors. Meanwhile, the phenotypic differences among individuals did not necessarily mean they must have sharp difference in genetic background as they met in the same group. Undoubtedly, these results aforementioned make this crop quite interesting to researchers for further investigating the molecular evolution of this special AABB group.     

Genetic Diversity of the Greater Yam (<Emphasis Type="Italic">Dioscorea alata</Emphasis> L.) and Relatedness to <Emphasis Type="Italic">D. nummularia</Emphasis> Lam. and <Emphasis Type="Italic">D. transversa</Emphasis> Br. as Revealed with AFLP Markers

Amplified fragment length polymorphism markers were used to assess the genetic relatedness between Dioscorea alata and nine other edible Dioscorea. These species include D. abyssinica Hoch., D. bulbifera L., D. cayenensis-rotundata Lamk. et Poir., D. esculenta Burk., D. nummularia Lam., D. pentaphylla L., D. persimilis Prain. et Burk., D. transversa Br. and D. trifida L. Four successive studies were conducted with emphasis on the genetic relationship within D. alata and among species of the Enantiophyllum section from Vanuatu. Study 1 was carried out to select a set of polymorphic primer pairs using 11 combinations and eight species belonging to five distinct sections. The four most polymorphic primer pairs were used in study 2 among six species of the Enantiophyllum section. Study 3 focussed mainly on the genetic relationship among 83 accessions of D. alata, mostly from Vanuatu (78 acc.) but also from Benin, Guadeloupe, New Caledonia and Vietnam. The ploidy level of 53 accessions was determined and results indicated the presence of tetraploid, hexaploid and octoploid cultivars. Study 4, included 35 accessions of D. alata, D. nummularia and D. transversa and was conducted using two primer pairs to verify the taxonomical identity of the cultivars `langlang', `maro' and `netsar' from Vanuatu. The overall results indicated that each accession can be fingerprinted uniquely with AFLP. D. alata is an heterogeneous species which shares a common genetic background with D. nummularia and `langlang', `maro' and `netsar'. UPGMA cluster analysis revealed the existence of three major groups of genotypes within D. alata, each assembling accessions from distant geographical origins and different ploidy levels. The analysis also revealed that `langlang', `maro' and `netsar' clustered together with the cultivar `wael' (D. transversa) from New Caledonia. Results are discussed in the paper.     

Relationship of European persimmon (Diospyros kaki Thunb.) cultivars to Asian cultivars, characterized using AFLPs

Sixty one persimmon (Diospyros kaki Thunb.) selections, including 17 Italian, 11 Spanish, 13 Japanese, six Korean, five Chinese, one Israeli, and eight of unknown origin, were evaluated for genetic differences by AFLP analysis. Relationships among cultivars were evaluated by UPGMA clustering, Neighbor Joining, and MultiDimensional Scaling. While similarities among groups were generally less than 0.60, both UPGMA and Neighbor Joining separated European and Asian cultivars. Spanish and Italian cultivars were not separated by any of the analyses, suggesting that they share a common gene pool, while Japanese, Chinese and Korean cultivars formed distinct clusters. Diversity within groups was greater than diversity between groups. Most cultivars were quite polymorphic (only 0.60–0.80 similarity between cultivars). In addition, the presence of several Japanese cultivars in the European group and a group of European cultivars nested between Chinese and Korean groups suggest that similar, but different progenitors were used in the development of the present European cultivars. ‘Kaki Tipo’ selections from different sources were clearly different by AFLP analysis, indicating that they are separate cultivars.     

Genetic relationships and diversity among Brazilian cultivars and landraces of common beans (Phaseolus vulgaris L.) revealed by AFLP markers

The genetic variation and relationships among 31 accessions of Phaseolus vulgaris L., and two representatives of Vigna unguiculata L., were evaluated by AFLP analysis. A total of 263 DNA fragments across all materials were scored using nine primer combinations, averaging 32 per primer. More than 95% of the amplification products showed polymorphism, indicating high variation at the DNA level among these accessions. Pair-wise genetic similarity (Jaccard's coefficient) ranged from 0.553 to 0.840, with a mean of 0.765. Twenty-three accessions (70%) clustered into three groups. A majority of the commercial cultivars (91%) clustered within a single group, whereas the landraces were distributed along all the variation. An apparent correlation with phaseolin types was detected. Results of this study suggest that Brazilian landraces truly represent the overall genetic variability of Phaseolus vulgaris, confirming the multiple origins of these materials, and their potential as a source of variation for breeding programs.     

AFLP and RFLP linkage map in <Emphasis Type="Italic">Coix</Emphasis>

Coix is a genus in the grass family placed in the tribe Maydeae. It is closely related to maize and is also used as a crop plant. Since many valuable traits have been identified recently in Coix, it is considered to be a valuable genetic resource, particularly for maize improvement. In this study, a Coix genetic linkage map was constructed using an F2 population of 131 individuals. Eighty AFLP and 10 RFLP markers were mapped, covering a total length of 1339.5 cM with an average interval of 14.88 cM. The map consisted of 10 linkage groups, were consistent with the chromosome numbers observed cytogenetically. Both AFLP and RFLP markers were used first for genetic analysis in Coix. AFLP markers were generated by two restriction enzyme combinations, EcoRI/MseI and PstI/MseI. A total of 1349 bands were amplified, of which 140 were polymorphic. The polymorphism detection efficiency of the two enzyme combinations was compared, and utility of AFLP markers to construct the linkage map was discussed. Ten RFLP markers detected by three different probes were distributed on eight different linkage groups. The results provide a foundation to map and isolate important genes in Coix, and to investigate its genomic architecture, possible origins, and relationship with maize at the DNA level.     

Optimum Sample Size for Estimating Gene Diversity in Wild Wheat using AFLP Markers

Genetic diversity in five wild types of wheat was estimated using Simpson's index (based on heterozygosity) applied to data from AFLP markers. For such studies, the cost of obtaining the required information increases both with the number of samples required to estimate diversity and with the number of markers used. When the population studied is in Hardy–Weinberg equilibrium (HWE), allelic frequencies follow the binomial expansion and parametric methods can be used to calculate the variance of the diversity index in terms of the number of individuals sampled. Inbred species are never in HWE. With regard to such populations, this study addresses the question of the sample size required to estimate gene diversity using a distribution-free re-sampling method. We studied populations of five wild species (Aegilops speltoides, Triticum urartu, Triticum boeoticum, Triticum dicoccoides, and Triticum araraticum) as sources of diversity. We used bootstrap re-sampling with varying sample sizes to develop a relationship between the precision of the diversity estimate and the sample size. Such a relationship was used to determine the samples required for capturing a given amount of diversity and its precision. We found that 5–6 samples are sufficient to obtain a standard error equal to 10% of the diversity in the populations of the species Ae. speltoides, T. dicoccoides and T. araraticum. However, more than 12 samples would be needed for populations of T. urartu and T. boeoticum. The procedure presented here can be used to obtain the optimum sample size for other crop species as well.