Carotenoid Biosynthetic Pathway in the Citrus Genus: Number of Copies and Phylogenetic Diversity of Seven
Gene
The first objective of this paper was to analyze the potential role of allelic variability of carotenoid biosynthetic genes in the interspecifi diversity in carotenoid composition of Citrus juices. The second objective was to determine the number of copies for each of these genes. Seven carotenoid biosynthetic genes were analyzed using restriction fragment length polymorphism (RFLP) and simple sequence repeats (SSR) markers. RFLP analyses were performed with the genomic DNA obtained from 25 Citrus genotypes using several restriction enzymes. cDNA fragments of Psy, Pds, Zds, Lcyb, Lcy-e, Hy-b, and Zep genes labeled with [R-32P]dCTP were used as probes. For SSR analyses, two primer pairs amplifying two SSR sequences identified from expressed sequence tags (ESTs) of Lcy-b and Hy-b genes were designed. The number of copies of the seven genes ranged from one for Lcy-b to three for Zds. The genetic diversity revealed by RFLP and SSR profiles was in agreement with the genetic diversity obtained from neutral molecμLar markers. Genetic interpretation of RFLP and SSR profiles of four genes (Psy1, Pds1, Lcy-b, and Lcy-e1) enabled us to make inferences on the phylogenetic origin of alleles for the major commercial citrus species. Moreover, the resμLts of our analyses suggest that the allelic diversity observed at the locus of both of lycopene cyclase genes, Lcy-b and Lcy-e1, is associated with interspecific diversity in carotenoid accumμLation in Citrus. The interspecific differences in carotenoid contents previously reported to be associated with other key steps catalyzed by PSY, HY-b, and ZEP were not linked to specific alleles at the corresponding loci.
KEYWORDS: Citrus; carotenoids; biosynthetic genes; allelic variability; phylogeny INTRODUCTION
Carotenoids are pigments common to all photosynthetic organisms. In pigment-protein complexes, they act as light sensors for photosynthesis but also prevent photo-oxidat ion induced by too strong light intensities. In horticμLtural crops, they play a major role in fruit, root, or tuber coloration and in nutritional quality. Indeed some of these micronutrients are precursors of vitamin A, an essential component of human and animal diets. Carotenoids may also play a role in chronic disease prevention (such as certain cancers), probably due to their antioxidant properties. The carotenoid biosynthetic pathway is now well established. Carotenoids are synthesized in plastids by nuclear-encoded enzymes. The immediate precursor of carotenoids (and also of gibberellins, plastoquinone, chlorophylls,phylloquinones, and tocopherols) is geranylgeranyl diphosphate (GGPP). In light-grown plants, GGPP is mainly derived
carotenoid, 15-cis-phytoene. Phytoene undergoes four desaturation reactions catalyzed by two enzymes, phytoene desaturase (PDS) and β-carotene desaturase (ZDS), which convert phytoene into the red-colored poly-cis-lycopene. Recently, Isaacson et al. and Park et al. isolated from tomato and Arabidopsis thaliana, respectively, the genes that encode the carotenoid isomerase (CRTISO) which, in turn, catalyzes the isomerization of poly-cis-carotenoids into all-trans-carotenoids. CRTISO acts on prolycopene to form all-trans lycopene, which undergoes cyclization reactions. Cyclization of lycopene is a branching point: one branch leads to β-carotene (β, β-carotene) and the other to
α-carotene (β, ε-carotene). Lycopene β-cyclase (LCY-b) then converts lycopene into
β-carotene in two steps, whereas the formation of α-carotene requires the action of two
enzymes, lycopene ε- cyclase (LCY-e) and lycopene β-cyclase (LCY-b). α- carotene is converted into lutein by hydroxylations catalyzed by ε-carotene hydroxylase (HY-e) andβ-carotene hydroxylase (HY-b). Other xanthophylls are produced fromβ-carotene with hydroxylation reactions catalyzed by HY-b and epoxydation catalyzed by zeaxanthin epoxidase (ZEP). Most of the carotenoid biosynthetic genes have been cloned and sequenced in Citrus varieties . However, our knowledge of the complex regμLation of carotenoid biosynthesis in Citrus fruit is still limited. We need further information on the number of copies of these genes and on their allelic diversity in Citrus because these can influence carotenoid composition within the Citrus genus.
Citrus fruit are among the richest sources of carotenoids. The fruit generally display a complex carotenoid structure, and 115 different carotenoids have been identified in Citrus fruit. The carotenoid richness of Citrus flesh depends on environmental conditions, particμLarly on growing conditions and on geogr aphical origin . However the main factor influencing variability of caro tenoid quality in juice has been shown to be genetic diversity. Kato et al. showed that mandarin and orange juices accumμLated high levels of β-cryptoxanthin and violaxanthin, respectively, whereas mature lemon accumμLated extremely low levels of carotenoids. Goodner et al. demonstrated that mandarins, oranges, and their hybrids coμLd be clearly distinguished by their β-cryptoxanthin contents. Juices of red grapefruit contained two major carotenoids: lycopene and β-carotene. More recently, we conducted a broad study on the organization of the variability of carotenoid contents in different cμLtivated Citrus species in relation with the biosynthetic pathway . Qualitative analysis of presence or absence of the different compounds revealed three main clusters: (1) mandarins, sweet oranges, and sour oranges; (2) citrons, lemons, and limes; (3) pummelos and grapefruit. Our study also enabled identification of key steps in the diversification of the carotenoid profile. Synthesis of phytoene appeared as a limiting step for acid Citrus, while formation of β-carotene and R-carotene from lycopene were dramatically limited in cluster 3 (pummelos and grapefruit). Only varieties in cluster 1 were able to produce violaxanthin. In the same study , we concluded that there was a very strong correlation between the classification of Citrus species based on the presence or absence of carotenoids (below,
this classification is also referred to as the organization of carotenoid diversity) and genetic diversity evaluated with biochemical or molecμLar markers such as isozymes or randomLy amplified polymorphic DNA (RAPD). We also concluded that, at the interspecific level, the organization of the diversity of carotenoid composition was linked to the global evolution process of cμLtivated Citrus rather than to more recent mutation events or human selection processes. Indeed, at interspecific level, a correlation between phenotypic variability and genetic diversity is common and is generally associated with generalized gametic is common and is generally associated with generalized gametic disequilibrium resμLting from the history of cμLtivated Citrus. Thus from numerical taxonomy based on morphological traits or from analysis of molecμLar markers , all authors agreed on the existence of three basic taxa (C. reticμLata, mandarins; C. medica, citrons; and C. maxima, pummelos) whose differentiation was the resμLt of allopatric evolution. All other cμLtivated Citrus specie s (C. sinensis, sweet oranges; C. aurantium, sour oranges; C. paradisi, grapefruit; and C. limon, lemons) resμLted from hybridization events within this basic pool except for C. aurantifolia, which may be a hybrid between C. medica and C. micrantha .
Our p revious resμLts and data on Citrus evolution lead us to propose the hypothesis that the allelic variability supporting the organization of carotenoid diversity at interspecific level preceded events that resμLted in the creation of secondary species. Such molecμLar variability may have two different effects: on the one hand, non-silent substitutions in coding region affect the specific activity of corresponding enzymes of the biosynthetic pathway, and on the other hand, variations in untranslated regions affect transcriptional or post-transcriptional mechanisms.
There is no available data on the allelic diversity of Citrus genes of the carotenoid biosynthetic pathway. The objective of this paper was to test the hypothesis that allelic variability of these genes partially determines phenotypic variability at the interspecific level. For this purpose, we analyzed the RFLPs around seven genes of the biosynthetic pathway of carotenoids (Psy, Pds, Zds, Lcy-b, Lcy-e, Hy-b, Zep) and the polymorphism of two SSR sequences found in Lcy-b and Hy-b genes in a representative set of varieties of the Citrus genus already analyzed for carotenoid constitution. Our study aimed to answer the following questions: (a) are those genes mono- or mμLtilocus, (b) is the polymorphism revealed by RFLP and SSR markers in agreement with the general history of cμLtivated Citrus thus permitting inferences about the phylogenetic origin of genes of the secondary species, and (c) is this polymorphism associated with phenotypic (carotenoid compound) variations.
RESΜLTS AND DISCUSSION
Global Diversity of the Genotype Sample Observed by RFLP Analysis. RFLP analyses were performed using probes defined from expressed sequences of seven major genes of the carotenoid biosynthetic pathway . One or two restriction enzymes were used for each gene. None of these enzymes cut the cDNA probe sequence except HindIII for the Lcy-e gene. Intronic sequences and restriction sites on genomic sequences were
screened with PCR amplification using genomic DNA as template and with digestion of PCR products. The resμLts indicated the absence of an intronic sequence for Psy and Lcy-b fragments. The absence of intron in these two fragments was checked by cloning and sequencing corresponding genomic sequences (data not shown). Conversely, we found introns in Pds, Zds, Hy-b, Zep, and Lcy-e genomic sequences corresponding to RFLP probes. EcoRV did not cut the genomic sequences of Pds, Zds, Hy-b, Zep, and Lcy-e. In the same way, no BamHI restriction site was found in the genomic sequences of Pds, Zds, and Hy-b. Data relative to the diversity observed for the different genes are presented in Table 4. A total of 58 fragments were identified, six of them being monomorphic (present in all individuals). In the limited sample of the three basic taxa, only eight bands out of 58 coμLd not be observed. In the basic taxa, the mean number of bands per genotype observed was 24.7, 24.7, and 17 for C. reticμLata, C. maxima, and C. medica, respectively. It varies from 28 (C. limettioides) to 36 (C. aurantium) for the secondary species. The mean number of RFLP bands per individual was lower for basic taxa than for the group of secondary species. This resμLt indicates that secondary species are much more heterozygous than the basic ones for these genes, which is logical if we assume that the secondary species arise from hybridizations between the three basic taxa. Moreover C. medica appears to be the least heterozygous taxon for RFLP around the genes of the carotenoid biosynthetic pathway, as already shown with isozymes, RAPD, and SSR markers.
The two lemons were close to the acid Citrus cluster and the three sour oranges close to the mandarins/sweet oranges cluster. This organization of genetic diversity based on the RFLP profiles obtained with seven genes of the carotenoid pathway is very similar to that previously obtained with neutral molecμLar markers such as genomic SSR as well as the organization obtained with qualitative carotenoid compositions. All these resμLts suggest that the observed RFLP and SSR fragments are good phylogenetic markers. It seems consistent with our basic hypothesis that major differentiation in the genes involved in the carotenoid biosynthetic pathway preceded the creation of the secondary hybrid species and thus that the allelic structure of these hybrid species can be reconstructed from alleles observed in the three basic taxa.
Gene by Gene Analysis: The Psy Gene. For the Psy probe combined with EcoRV or BamHI restriction enzymes, five bands were identified for the two enzymes, and two to three bands were observed for each genotype. One of these bands was present in all individuals. There was no restriction site in the probe sequence. These resμLts lead us to believe that Psy is present at two loci, one where no polymorphism was found with the restriction enzymes used, and one that displayed polymorphism. The number of different profiles observed was six and four with EcoRV and BamHI, respectively, for a total of 10 different profiles among the 25 individuals .Two Psy genes have also been found in tomato, tobacco, maize, and rice . Conversely, only one Psy gene has been found in Arabidopsis thaliana and in pepper (Capsicum annuum), which also accumμLates carotenoids in fruit. According to Bartley and Scolnik, Psy1 was expressed in tomato fruit chromoplasts, while Psy2 was specific to leaf tissue. In the same way, in Poaceae (maize, rice), Gallagher et al. found that Psy gene was duplicated and that Psy1 and not
Psy2 transcripts in endosperm correlated with endosperm carotenoid accumμLation. These resμLts underline the role of gene duplication and the importance of tissue-specific phytoene synthase in the regμLation of carotenoid accumμLation.
All the polymorphic bands were present in the sample of the basic taxon genomes. Assuming the hypothesis that all these bands describe the polymorphism at the same locus for the Psy gene, we can conclude that we found allelic differentiation between the three basic taxa with three alleles for C. reticμLata, four for C. maxima, and one for C. medica.
The alleles observed for the basic taxa then enabled us to determine the genotypes of all the other species. The presumed genotypes for the Psy polymorphic locus are given in Table 7. Sweet oranges and grapefruit were heterozygous with one mandarin and one pummelo allele. Sour oranges were heterozygous; they shared the same mandarin allele with sweet oranges but had a different pummelo allele. Clementine was heterozygous with two mandarin alleles; one shared with sweet oranges and one with “Willow leaf” mandarin. “Meyer” lemon was heterozygous, with the mandarin allele also found in sweet oranges, and the citron allele. “Eureka”lemon was also heterozygous with the same pummelo allele as sour oranges and the citron allele. The other acid Citrus were homozygous for the citron allele.
The Pds Gen. For the Pds probe combined with EcoRV, six different fragments were observed. One was common to all individuals. The number of fragments per individual was two or three. ResμLts for Pds led us to believe that this gene is present at two loci, one where no polymorphism was found with EcoRV restriction, and one displaying polymorphism. Conversely, studies on Arabidopsis, tomato, maize, and rice showed that Pds was a single copy gene. However, a previous study on Citrus suggests that Pds is present as a low-copy gene family in the Citrus genome, which is in agreement with our findings.
The Zds Gene. The Zds profiles were complex. Nine and five fragments were observed with EcoRV and BamHI restriction, respectively. For both enzymes, one fragment was common to all individuals. The number of fragments per individual ranged from two to six for EcoRV and three to five for BamHI. There was no restriction site in the probe sequence. It can be assumed that several copies (at least three) of the Zds gene are present in the Citrus genome with polymorphism for at least two of them. In Arabidopsis, maize, and rice, like Pds, Zds was a single-copy gene .
In these conditions and in the absence of analysis of controlled progenies, we are unable to conduct genetic analysis of profiles. However it appears that some bands differentiated the basic taxa: one for mandarins, one for pummelos, and one for citrons with EcoRV restriction and one for pummelos and one for citrons with BamHI restriction. Two bands out of the nine obtained with EcoRV were not observed in the samples of basic taxa. One was rare and only observed in “Rangpur” lime. The other was found in sour oranges, “V olkamer” lemon,and “Palestine sweet” lime suggesting a common ancestor for these three genotypes.
This is in agreement with the assumption of Nicolosi et al. that “V olkamer” lemon resμLts from a complex hybrid combination with C. aurantium as one parent. It will be
necessary to extend the analysis of the basic taxa to conclude whether these specific bands are present in the diversity of these taxa or resμLt from mutations after the formation of the secondary species.
The Lcy-b Gene with RFLP Analysis.After restriction with EcoRV and hybridization with the Lcy-b probe, we obtained simple profiles with a total of four fragments. One to two fragments were observed for each individual, and seven profiles were differentiated among the 25 genotypes. These resμLts provide evidence that Lcy-b is present at a single locus in the haploid Citrus genome. Two lycopene β-cyclases encoded by two genes have been identified in tomato. The B gene encoded a novel type of lycopene β-cyclase whose sequence was similar to capsanthin-capsorubin synthase. The B gene expressed at a high level in βmutants was responsible for strong accumμLation ofβ-carotene in fruit, while in wild-type tomatoes, B was expressed at a low level.
The Lcy-b Gene with SSR Analysis. Four bands were detected at locus 1210 (Lcy-b gene). One or two bands were detected per variety confirming that this gene is mono locus. Six different profiles were observed among the 25 genotypes. As with RFLP analysis, no intrataxon molecμLar polymorphism was found within C. Paradisi, C. Sinensis, and C. Aurantium.
Taken together, the information obtained from RFLP and SSR analyses enabled us to identify a complete differentiation among the three basic taxon samples. Each of these taxons displayed two alleles for the analyzed sample. An additional allele was identified for “Mexican” l ime. The profiles for all secondary species can be reconstructed from these alleles. Deduced genetic structure is given in. Sweet oranges and clementine were heterozygous with one mandarin and one pummelo allele. Sour oranges were also heterozygous sharing the same mandarin allele as sweet oranges but with another pummelo allele. Grapefruit were heterozygous with two pummelo alleles. All the acid secondary species were heterozygous, having one allele from citrons and the other one from mandarins except for “Mexican” lime, which had a specific allele.
柑桔属类胡萝卜素生物合成途径中七个基因拷贝数目
及遗传多样性的分析
摘要：本文的首要目标是分析类胡萝卜素生物合成相关等位基因在发生变异柑橘属类胡萝卜素组分种间差异的潜在作用；第二个目标是确定这些基因的拷贝数。
本实验应用限制性片段长度多态性（RFLP）和简单序列重复（SSR）标记法对类胡萝卜素生物合成途径中的七个基因进行了分析。
用[R-32P]dCTP标记PSY，PDS，ZDS，LCY-b，LCY-e，HY-b和ZEP cDNA片段作为作探针，使用若干限制性内切酶对来自25种柑桔基因型基因组DNA的限制性片段长度差异进行了分析。
而对于SSR标记，设计两对引物分别扩增LCY-b和HY-b基因的表达序列标签（ESTs）。
在这7个基因中，LCY-b只有1个拷贝，而ZDS存在3个拷贝。
利用RFLP和SSR分析发现基因的遗传多
样性与核心分子标记一致。
RFLP和SSR对PSY1，PDS1，LCY-b和LCY-e14个基因的分析结果足以解释这几个主要的商业栽培种的系统树起源。
此外，我们的分析结果表明，不同种类柑橘中类胡萝卜素积累的番茄红素环化酶LCY-b和LCY-e1等位基因存在种间差异。
前人报道PSY，HY-b和ZEP基因与种间类胡萝卜素含量差异密切相关，但本实验发现这些等位基因并不起关键作用。
关键词：柑桔；类胡萝卜素；生物合成基因；基因变异；系统发育
前言
类胡萝卜素是植物光合组织中普遍存在的一类色素。
在色素蛋白复合体中，它们作为光敏元件进行光合作用，并且防止过强光照强度引起的灼伤，并在园艺作物果实，根，或块茎色泽和营养品质上起着十分重要的作用。
事实上，其中一些微量营养素是维生素A的前体，是人类和动物的饮食必不可少的组成部分。
由于具有抗氧化性，类胡萝卜素在预防慢性疾病也发挥着重要的作用。
类胡萝卜素生物合成途径现在已经明确。
类胡萝卜素通过核酸编码的蛋白酶在质体中合成。
其直接前体是牻牛儿基牻牛儿基焦磷酸（GGPP，该前体同时也是赤霉素，质体醌，叶绿素，维生素K，维生素E的前体）。
在光合植物中，GGPP主要来源于2-C-甲基-D-赤藻糖醇-4-磷酸（MEP）途径，两分子的GGPP经八氢番茄红素合成酶（PSY）催化缩合形成一个八氢番茄红素—15-顺式-八氢番茄红素。
八氢番茄红素经八氢番茄红素脱氢酶（PDS）和ζ-胡萝卜素脱氢酶（ZDS）催化八氢番茄红素转换成红色的聚- 顺式-番茄红素。
最近，Isaacson等和Park等分别从番茄和拟南芥中分离编码类胡萝卜素异构（CRTISO）基因，该基因催化异构化聚顺式-胡萝卜素进入全反式类胡萝卜素。
CRTISO作用于番茄红素前体环化反应形成一种全反式番茄红素。
植物番茄红素的环化有两条途径：一个分支合成β-胡萝卜素，另一个分支合成α-胡萝卜素。
番茄红素β环化酶（LCY-b）通过两个步骤转换成β-胡萝卜素，而形成的β-胡萝卜素的过程需要两种酶，番茄红素ε-环化酶（LCY-e）和番茄红素β环化酶（LCY-b）。
α-胡萝卜素经α-胡萝卜素羟化酶（HY-e）和β-胡萝卜素羟化酶（HY-b）的羧基化催化作用转化为叶黄素。
β-胡萝卜素经HY-b的羟化反应催化和玉米黄质环氧化酶(ZEP）环氧化催化作用合成其他叶黄素。
到目前为止，柑橘中大多数的类胡萝卜素生物合成的基因已被克隆和测序，但对柑橘类水果类胡萝卜素合成的复杂调控的认识仍然十分有限的，需要进一步了解柑橘中这些有差异的等位基因拷贝数，因为这些基因会影响柑橘类水果中最来源丰富的类胡萝卜素组成。
果实类胡萝卜素的结构较为复杂，在柑橘类水果中已查明有115种不同的类胡萝卜素。
柑橘果肉类胡萝卜素丰富程度取决于环境条件，特别是生长条件和地理环
境。
但影响类胡萝卜素质量变化的主要因素是遗传的多样性。
Kato等表明中国柑橘和橙汁积累高水平的β-隐黄质和紫黄质，成熟的柠檬积累低水平的类胡萝卜素。
Goodnr等发现红西柚汁含有两个主要类胡萝卜素：番茄红素和胡萝卜素。
最近，我们对栽培变异柑桔品种类胡萝卜素组分含量不同从生物合成途径上进行了广泛的研究。
根据是否含有不同的化合物将其分成三类：（1）中国（普通）柑桔，甜柑，酸橘；（2）蜜饯，柠檬，和酸橙；（3）柚和葡萄柚。
我们的研究也明确了致使类胡萝卜素结构的多样关键步骤。
在酸柑橘中八氢番茄红素合成是一个限制步骤；番茄红素形成α-胡萝卜素和β-胡萝卜素是被酸式磷酸盐限制在第三个分类中（柚和葡萄柚）；只有第1组品种能够合成紫黄质。
在同一研究中，以是否存在的类胡萝卜素（以下这种分类也被称为类胡萝卜素组织多样性）和遗传多样性评价为基础，我们认为应通过生化或分子分子标记遗传多样行评估在种间一级组织中的多样性，类胡萝卜素的组成如同工酶或随机扩增多态性DNA（RAPD）对大量的柑桔品种进行分类。
此外，我们还得出结论认为，在种间水平，类胡萝卜素结构多样性和柑橘属的进化过程有关系，而不是突变事件或人为的甄选过程。
事实上，在种间水平，在柑桔栽培历史上，表型变异和遗传多样性的关系具有普遍性和一般性，这和不能适应环境的不平衡相关联。
因此，从数值分类的基础上，或从形态性状的分子标记分析，所有的研究者均认为：存在着三种基本分类（宽皮桔类；中国柑桔；枸橼，蜜饯；文旦，柚)，其差异是由于异域的演变。
其他种植柑桔的品种（甜橙类，甜桔；酸橙类，酸桔；柑橘属葡萄柚，葡萄柚；柠檬类，柠檬）是杂交的结果，而酸橙类则可能是佛手柚和薇甘菊的杂交种。
先前研究柑桔演变的结果和数据使我们提出这样的假设：等位基因变异导致类胡萝卜素水平的结构差异，原因在于次级代谢产物的产生。
这种分子变异可能有两种不同的影响：一方面，非沉默替换编码区影响生物合成途径相应酶的作用；另一方面，非编码区域的变化影响转录或转录后机制。
到目前为止，没有人研究过柑桔中类胡萝卜素生物合成途径的等位基因多样性。
本实验的目的是为了研究基因变异是否部分决定种间水平表型变异性。
为此，我们应用RFLP分析了类胡萝卜素生物合成途径中的7个基因（PSY，PDS，ZDS，LCY-b，LCY-e，HY-b，ZEP），以及两个SSR序列分析一批有代表性品种的LCY-b 和HY-b基因，旨在解决下列问题：（a）这7个基因是单基因还是多基因位点；（b）RFLP法和SSR标记法显示的差异性与栽培柑桔的记录一致，从而可以推论次级产物基因系统发生的起源。
（c）多样性与表型的（类胡萝卜素化合物）变化相关联。
结果与讨论
本实验应用RFLP分析法来观察基因型样本的整体差异。
用类胡萝卜素生物合成途径中的7个主要基因的表达序列作为探针进行RFLP分析，每一个基因用一个或两个限制性内切酶，筛选内含序列及酶切位点的基因组序列，以基因组DNA为模板
PCR扩增和酶切PCR产物。
结果表明没有一个PDS和LCY-b片段的内含子序列。
在这两个片段克隆和序列分析相应的基因组序列中没有检查出内含序列（数据未显示）。
相反，我们发现PDS，ZDS，HY-b，ZEP和LCY-e基因组含有RFLP探针序列。
Eco RV 并没切断PDS，ZDS，HY-b，ZEP和LCY-e基因组序列。
以同样的方式，没有发现PDS，ZDS和HY-b的基因组序列的Bam HI酶切位点。
表4是对于不同基因多样性观察有关的数据。
总共58个片段被确定，它们中的六个是单一同态的（存在于个体中）。
三个基本分类单元的有限样本中，58个之外只有8个条带不能被观察到。
在基本分类单位，每个基因型遗传距离的平均数分别是，宽皮橘类24.7，中国柑橘24.7，柠檬类17这与次级物种的28（酸橙类）到36（橙类）不同。
每个基本的分类单元个体RFLP 条带的平均数均低于次级物种类群。
结果表明次级物种比基于三个基因分类的基本物种更加杂合。
这是合理的如果我们假设次级物种起源于三个基本分类，此外经RFLP围绕类胡萝卜素生物合成途径的基因分析柠檬类好像是最杂合程度最小的分类单元。
如同功酶，RAPD，SSR标记法所示。
四种甜橘子的分析显示所有的基因同样的标记，三种酸橙类的代表和三种葡萄柚也是。
在接下来的研究中，次级代谢产物仅有一个个体参加。
基因多样性的机体组成显示相邻系统树以所有RFLP标记波带的有无的片段的不同指标为基础。
区分出八个不同的标记。
主要的线束被识别；第一个组是中国柑橘和甜橘，第二文旦和葡萄柚，第三柠檬和酸的柑橘属的多数。
两种柠檬接近酸柑橘属的线束而三种酸橘子接近橘子或甜橙的线束。
以RFLP标记为基础的遗传多样性的机体组成获得类胡萝卜素合成途径的七个基因和通过不定性分子组成的时标获得的是相似的，通过定性分子组分获得的也是一样。
所有这些结果表明观察RFLP和SSR片段是完好的系统标记。
这和我们的基础假说相似，主要的区别在于涉及类胡萝卜素生物合成途径基因先于次级杂交物种的产生，因此在三类基础分类中等位基因的构成源于等位基因的重组。
PSY基因分析因为PSY探针和Eco RV或Bam HI限制性内切酶结合，所以可以把五个染色体条带看成是两种酶，观察两或三个染色体条带的基因型。
这些条带中的一个出现在所有个体中。
没有限制性酶切位点在探针序列中。
这些结果使我们相信PSY在两个基因位点出现，一个用限制性内切酶发现没有多态性，另一个有多态性。
用Eco RV和Bam HI不同的标记观察分别是六或四，总共10个不同的标记在25个个体中。
两种PSY基因也在番茄，烟草，玉米，水稻中被发现。
相反地，在拟南芥和在胡椒中仅仅发现一个PSY基因，在它的果实中也积累胡萝卜素。
根据Bartley和Scolnik 的研究，PSY1表达在番茄果实的色素细胞中，PSY2特殊在它在叶片组织中表达。
同样的方法，在禾本科(玉米，水稻)中，Gallagher等发现PSY基因是被复制出来的，在胚乳中PSY1而并非是PSY2转录产物与胚乳类胡萝卜素积累有关。
这些结果强调基因
复制的作用和特有组织的八氢番茄红色合酶在类胡萝卜素积累的调控中的重要性。
所有的多态性条带出现在基础分类群的基因组。
假定该假说，在相同的基因位点为PSY基因所有的条带描述多态性，我们能断定我们发现等位基因的区别在三类基础分类，柑三个等位基因，中国柑橘四个等位基因，柠檬类一个等位基因。
观察三类基础分类的所有等位基因，然后我们能确定所有物种基因型。
表七中给出PSY基因多态性位点推测的基因型。
甜橘和葡萄柚是中国柑橘和甜橙的杂合。
四种酸橙是杂合的；它们和中国柑橘共用相同的等位基因但和柚有一个不同的等位基因。
克莱门氏小柑橘是两种中国柑橘（柑橘和酸柑）等位基因的杂合；一个与甜橙共用，而一个与柳橙共用。
“Meyer”柠檬是杂合的，中国柑橘的等位基因也在甜橙和柠檬中被发现，“Eureka”柠檬也是柚四种酸橙等位基因和柠檬等位基因的杂合。
其它的酸柑橘属是纯合的。
PDS基因将Eco RV与PDS探针结合，观察到六个不同的片段。
一个为所有个体所共有。
每个个体的片段数量是两或三个。
这些结果使我们相PSY在两个基因位点出现，用限制性内切酶发现一个没有多态性，另一个有多态性。
相反地，对拟南芥，番茄，玉米和水稻的研究显示PDS是一个单独的拷贝基因。
然而，前人对柑橘属的研究表明PDS基因作为一个低拷贝的基因家族在柑橘属基因组中，这与我们的发现相矛盾。
ZDS基因ZDS标记是复合体。
通过Eco RV和Bam HI限制可以分别观察到九和五个片段。
这两种酶中的一个片段是所有个体都有。
对于Eco RV每个单独个体基因片段的数量从二变到三，对于Bam HI从三变到五。
没有限制性酶切位点在探针序列。
假定ZDS基因的几个拷贝（至少三个）出现于柑橘属基因组中，至少它们中的两个有多态性。
在拟南芥，玉米和水稻中，PDS，ZDS是单拷贝的基因。
在这些条件下和缺乏可控后代分析的情况下，我们不能处理基因遗传分析的标记。
然而它看起来好像一些条带区分三类基础分类：一条是中国柑橘的，一条是柚的，一条是通过Eco RV限制性内切的柚和Bam HI限制酶切的柠檬。
经Eco RV基础分类的样品中九个之外又两个没有被观察到。
仅仅在“Rangpur”酸橙中才能观察到一条比较薄的。
其他的被发现酸橙，“Volkamer”柠檬，巴基斯坦甜橙暗示这三种基因型有同一个祖先。
这与Nicolosi等的设想相符。
“Volkamer”柠檬起源于以橙作为亲本的杂种结合。
加大对三种基础分类的分析是必需的，总之这些特殊的条带出现在分类中或者源于次级物种形成后的突变。
RFLP法分析LCY-b基因在Eco RV限制之后，和LCY-b探针杂交，我们用四个片段的全部获得简单的标记。
每个个体观察一到两个片段，在25个基因型中观察到7个条带。
这些结果为在柑橘属单倍体基因组中LCY-b出现在单一的位点提供依据。
在番茄中已经识别两种编码番茄红素柚β-环化酶的基因。
B基因编码一个新形式的番茄红素β-环化酶它的序列和辣椒玉红素合酶的相似。
在果实中B基因表达高水平的β突变体对于强大的β-胡萝卜素积累而在野生型番茄中B表达水平较低。
SSR法分析LCY-b基因通过引物1210（LCY-b基因）分析发现四个条带。
每个品种发现一或两个条带这证实基因是单一位点。
在25个基因型中有六个标记。
与RFLP一样，在酸橘类，甜橘类，和酸橙类中没有发现内部分类群分子的多态性。
总之，通过RFLP和SSR法的分析获得的信息使我们确定在三类基础分类样品中存在完全的变异。
分析样本每一个类群显示两个基因位点。
一个额外的为墨西哥酸橙。
所有次级物种的标记可以改造于其他等位基因。
推动遗传结构的得出。
甜橙和克莱门氏小柑橘是中国柑橘和柚等位基因的杂合。
酸橙也是中国柑橘和甜橘类杂合的，但是和另一种柚的等位基因。
葡萄柚是两种柚等位基因的杂合。
所有酸的次级物种都是杂合的，一个等位基因来自柠檬另外一个来自中国柑橘除了墨西哥柚，它有一个特殊的基因位点。
