Illustrative explanation of Genetics behind Marker Assisted Backcross breeding (MABC)

Concept of Maintenance Breeding

Concept of maintenance breeding in cotton

Maintenance breeding is a branch of breeding, implemented to maintain the genetic purity, vigor, yield potential and adoptation of a variety or a parent. There are numerous examples of varieties lost their yield potential due to the lack of maintenance breeding (Peng.  2010). The importance of maintenance breeding can be easily understood by investigating the genetic cause behind the loss of yield potential.

Cotton is the often cross pollinated crop with predominance of self pollination and low amount of (~5% ) cross pollination. This type of pollination system has two important features. In  open pollination, there will be a free exchange of pollens between plants. Secondly, Expression of heterosis (increase of hybrid vigor due to the heterozygosity of the Hybrid). The different alleles of the same gene contributes more to the phenotype at heterozygous state than at homozygous due to over dominance of alleles.  The genes at homozygous state leads to inbreeding depression or loss of hybrid vigor.

The genetics of an open pollinating pureline

A variety is a ‘pure line’. The progenies of a pureline are homozygous and homogenous in nature. Seed multiplication in cotton is taken at isolation fields  and maintained by open pollination. Though this population is said to be homozygous and homogenous, this is not the actual case.

The genetic makeup of an open pollinating variety will be

• Homozygous for morphological/qualitative traits
• Heterozygous for agronomical/Quantitative traits

The qualitative characters are controlled by one or few genes, and quantitative traits are controlled by polygene. The varietal development breeding involves crossing of two parents followed by subsequent generations of selection and selfing till the selected line attains homozygosity. The homozygous progeny is  now a pureline.   The homozygosity of qualitative genes leads to uniformity in the morphological traits. At the same time the agronomical traits suffers  inbreeding depression due to the homozygosity in polygenic alleles.

When the variety is brought to the open pollination from selfing, the variety regains its vigor in one cycle of open pollination. This is evident from the increased vigor of the progenies of the open pollinated variety. This clearly indicates the heterozygosity in polygenic traits are important for the maintenance of ‘varietal heterosis’ for vigor and yield potential.Out crossing in cotton is reported occur at the proportion of ~5%. Most of the out crossings occur within one meter of distance. Hence each plant receives ~5% pollen from the nearby plants leads to the heterozygosity of quantitative loci.
The  Genotype x  environment (G x E)  is the  another important characteristic of polygenic traits.  The environment influences more than 30% of the phenotypic expression of polygenes. Progenies of a pureline will differ in the G x E interaction ability. To put it in simple, consider, if two plants has same genotype, grown under same environment, the two plants might not have the same interaction with the environment, and the ‘fittest’ plants potential will be little more than the other plant.

What happens when there is no ‘Maintenance Breeding’?

Loss of genetic purity
Genetic purity is uniformity of plants in terms of morphological traits. Any variation in the qualitative traits indicates disturbance of genetic purity. In my previous blog, I have explained the appearance of such ‘off type’ plants in the seed multiplication plot. Heterozygosity, outcrossing, and point mutation are agents, creating variation in a pure line. If the variations are un noticed over the generations, result in the amplification of off type plants, and loss of genetic purity

Loss of vigor and potential
As we mentioned earlier, heterozygosity at loci of agronomical traits are important for maintaining the vigor. Under continuous selfing, the variety suffers inbreeding depression. The open pollination restores the heterosis or vigor of that variety due to some extend of outcrossing with nearby plants. But outcrossing is very limited in cotton and self-pollination is predominant. This amount of pollen exchange is not sufficient to get desired heterozygosity to retain the yield potential of the variety. Without maintenance breeding, the variety will approach the homozygous state even under open pollination, causes inbreeding depression

Loss of fitness/adaptation
Genotype x Environmental interactions is the measure of a genotypes ability to adopt for the changing environmental conditions. The environmental conditions changes over the seasons. The temperature, drought and salinity increases every year, and it has negative impact on a genotypes vigor and potential. If the variety in not maintained under changing environments, the relative fitness of the variety will get drastically reduced.

How maintenance breeding is done

The maintenance of a variety requires maintenance of genetic purity, vigor and fitness separately but altogether in the same plot.

Maintaining genetic purity:
The visible deviations in the morphological traits can be easily identified by the careful inspection of the plot. The off type plants should be immediately removed from the field to avoid the out crossing with nearby ‘pure’ plants. If the off type plants proportion is less than 0.01%, the removal of those plants is enough to maintain the genetic purity. Because the off type plants might be the results of either natural out crossing or by mutation. If the off type proportion is more than 0.1%, indicates the heterozygosity of few plants in the source seeds. In this case removal of off type plants are not enough, since some plants will be at heterozygous and escapes the visible detection of off phenotypes. In such situations, the genetic purity can be maintained by selection of  pure plants form the plot and selfing of the plants. The selfed seeds are raised in the next season in plant to row method. The  plants of a segregating progenies are removed from the plot and only the non-segregating uniform progenies are retained and allowed for open pollination.

Maintenance of vigor and yield potential

The inbreeding depression due to the preponderance of self-pollination can be alleviated by sib mating of individual plants. The best performing plants are selected and crossing is performed between all selected plants in all combinations. Each plant is used as a male as well as female to restore the cytoplasm of the all selected plants. Sib crossing ensures the union of alleles of different plants. The progenies of sib mated plants expresses the heterosis in vigor due to the higher level of heterozygosity in the quantitative loci

Maintenance of fitness
To maintain the fitness of a variety, it should be routinely brought in to the environment and perfoamce is monitored. The variety maintained by proper roguing and sib mating, might have high genetic purity  and uniform vigor but differs in fitness levels. productivity of some plants are superior to the other plants due to positive G x E interactions. Selection of superior plants increases the adoptability of the genotypes to that particular environment. The important point to note here is, selection will not improve the superiority of the genotypes. (“Selection is not effective within a pure line”. Johansen. 1906). Selection is only effective for the retention of ‘superiority’.  When the best performing plants are selected under particular environmental conditions, those plants will express more fitness in the successive generations of varietal Maintenance. To increase the longevity of the fitness, the variety should be maintained once in two years under the particular environmental conditions and best interacting (G x E) plants  should be selected as the source for next round of multiplication.

Procedure for Maintenance Breeding

One cycle of maintenance breeding involves

Year 1
Raising the variety in isolation fields under open pollination, rouging of off type plants, selection of best plants and sib mating between the selected plants

Year 2
Raising the sib progenies, rouging of off type plants, and selection of best plants.

After one or two years, the cycle should be started again. The selected plants at the end of year 2 will be the source for the next cycle.

Illustrations of outcrossing and sibmating in Cotton

Genetics of ‘off type’ plants in seed multiplication plots of cotton (Gossypium hirsutum L.)

This blog is about how to interpret the appearance of ‘off type’ plants in foundation seed progenies, regardless of the 100% purity in the breeder seed plots

Breeder seed is the base source for the quality hybrid seed production in cotton or for any crops. Foundation seeds are multiplied from breeder seed  and used as the source seed for the certified seed production. To maintain the higher genetic purity standards at the certified seeds, the genetic purity of breeder seed  is very important.

Despite of the genetic purity and uniformity of plants at breeder seed, at rare instances there is a distortion of genetic purity at the foundation seed multiplication plots. There will be a chance for two kinds of 'off type' plants that  appears in foundation plots.

1. Plants of different variety of same species. This is due to admixture of seeds during seed processing steps and can be eliminated by stringent quality control checks.
2. Plants of same variety of same species with one or two DUS characters altered. This kind of off types are more interesting to breeders since those plants are not appeared on the breeder seed plot. Based on the DUS character altered and proportion of such off type plants, breeder might able to give a genetic explanation for the deviation of purity

Here we will investigate the situations in which, ithe breeder seed plot is morphologically 100% pure, but segregation and appearance of off types at foundation field arises. Based on the proportion of the off type plants, the genetic cause can be explained. We are not taking account of admixture plants into our discussion, because the occurrence of admixture is purely on physical means, and no genetics involved.

1.) 10% off type plants (10/100) plants with altered DUS characters

This is very rare situation, arises due to segregation at large loci in some of the plants. For some breeding lines the fixation of homozygosity takes more generations of selfing. Though the progenies of breeding lines looks homogenous, some plants might still be heterozygous at some loci. When such lines are advanced to breeder seed multiplication, (owing to the commercial success of the hybrid) the heterozygous plants escapes detection. When advanced to foundation seed multiplication, some plants appear as distinct from the original parent. The escape  from breeder seed plot is  due to heterozygous and dominanace of genes. During the pollination at breeder seed multiplication, selfing occurs at great extend, leads to fixation of some  genes in the progenies of the heterozygous plants. The genotype of the progenies is now distinct from the original parent and appears as ‘off type’ in the foundation field.

2.) 1 % of off type plants (1/100) plants with altered DUS characters

In cotton, some of the DUS characters like petal colour, pollen colous are coded by single major genes. The alleles of the gene has alternate phenotypes. If the trait is encoded by a dominant gene, the homozygous and the heterozygous plants will express one phenotype and the recessive plants will express the alternate phenotype. If the breeder seed plot is rich in heterozygous, and dominant homozygote plants, all the plants will be phenotypically similar. The heterozygous plants segregates and produces recessive genotype progenies, that appears as alternate ‘off type” at foundation fields.

3.) 0.1% of off type plants (1/1000) plants with altered DUS characters

The above explanation holds true for this proportion of plants, except the fact that initial load of heterozygous plants at breeder seed plots will be lesser than above case. If there are one  or few heterozygous plants at breeder seed plots, this will lead to production of 0.1% of plants with recessive phenotype.

4.) 0.01% of plants (1/10,000) plants with altered DUS characters

So far heterozygosity can be accounted for the production of off type plants. But Hetorozygosity cannot explain the appearance of 0.01% of ‘off type’ plants because, a single heterozygous plant is enough to produce 0.1% of off type plants at foundation stage. Even if the breeder seed plot is 100% genetically pure, the appearance of such proportions of off types are possible and needs an investigation. In cotton, the appearance of 1/10,000 plants off types can be attributed to natural out crossing by honey bees or other insect pollinators. The out crossed plants will be of intermediate phenotype, carries few altered DUS trait. Hutmacher (2006) pointed out that 0.04% of out crossing occurs at up to 1500 distance with insect pollinators in cotton commercial fields. The cotton pollen is heavy and sticky in nature and out crossing through wind never exceeds  more than few meters

5.) 0.001% of plants (1/1,00,000) plants with altered DUS characters

Insect pollination can also explain 0.001% of plants could be the result of out crossing. The out crossed plants appear as intermediate phenotype of original parent and the pollen donar.  In very rare instance, the off type plants are same as original parents, except for the one DUS character differ from original parent. Classical genetics cannot explain appearance of such type plants at this 0.001% proportions. In the absence of segregation and outcrossing,  the appearance of 1/10, 00,000  off type plants can be explained by the population genetics

The seed multiplication plot in isolation is equivalent to population of random mating individuals. According to the Hardy Weinberg law, In a random mating population, the gene and the genotype frequencies are remain constant through generation to generations, unless selection, migration or mutation operates on the population. In a breeder seed multiplication plots no selection is practiced since all plants are homogenous and homozygous. The isolation distances eliminate the possibility of population migration through out crossing. Mutation could be the only agent acting on the seed multiplication population. The average mutation rate of living organism is 1/10, 00,000 per gene per gamete. Thus, one out 10, 00,000 pollens will carry a mutation in any one of the gene. A single cotton flower produces 30,000 pollens. Therefore one pollen in every three flowers might carry a mutation in a gene. But most of the mutated genes does not code for visible phenotype and undetectable at progenies. If the mutation occurs at the genes codes for the any one of the DUS characters, the mutation can be detected in the progenies as off types, at the proportions 1/10,00,000 to 1/1,00,00,000.

Difference between gene and marker

Young graduates often confused with the exact difference between gene and marker. The definition for gene and markers could be found in 100's of text books and websites. But nowhere the difference between them is mentioned. I will put a simple explanation for differentiating gene from markers.

Genes and markers are segments of DNA reside on chromosomes. Gene is a coding part of the DNA , encodes protein or RNA molecules which has specific function in the cells. The phenotypic traits of an organism are corresponding to particular genes.  Molecular tools has identified thousands of genes, and each gene was assigned to a particular function. But the sequence information and locations of the genes on chromosomes are very less studied and for most of the genes  the information is not available. Genetic mapping and physical mapping studies has been carried out to locate the genes on chromosomes. This mapping studies are limited to only those genes which has visible phenotypic trait. Irrelevant of the location, the genes encodes a function.

Markers which have no specific functional expression in cells as like genes. They are DNA segments which sequence is fully or partly known and  the relative location of the markers on chromosome is known. Genetic mapping studies  located markers on a specific chromosomes and estimated the relative distance between the markers and to the markers and genes. A gene with known function and unknown location can be tagged with a marker with known location if the gene  resides close to the marker on the chromosomes. If the marker position on the chromosome is unknown, those markers cannot be used for genetic mapping or breeding programs. They can be used only for diversity studies.

To generalize the above discussion, a gene has function but the sequence and location on the chromosome is mostly unknown. The marker has known sequence information and mapped on a chromosome, but do not have any functional role. This definition is only a generalization and not applicable to all genes and markers.

When a gene qualifies as a marker?

If the sequence of the gene and its location on the chromosome is known, that gene can be used as the marker for genetic s studies as like markers.

Do not confuse the genic markers with marker genes. Marker genes encodes a functional protein which can be detected or assayed by experiments and being used in the cytological studies and transgenic developments.

Why recurrent plant is always used as a female parent in backcross breeding programmes?

Answer:

The basic objective of back cross breeding program is to transfer one or two genes from a donar plant to a well adopted recurrent cultivar. Here the term 'adopted' means, adaptation of the cultivar to the wide environmental conditions. The back cross process aims to recover as much of recurrent parent genome is subsequent back crosses in addition to the donar genes of interest. 

There are two reason for using the recurrent cultivar as female parent. First reason is the recurrent parent factor. The fertilization process involves fusion of pollen nucleus and ovule nucleus. Pollens carries only the nucleus from the pollen mother cell. The the cytoplasm of pollen cells will be degraded during the maturity of the pollens. On the other hand, the ovule cells carries entire cytoplasm along with nucleus of the ovule mother cells. During zygote formation, pollen nucleus (n) and ovule nucleus (n) fuses and develops into zygote (2n) but the cytoplasmic content of female is remain undisturbed. The developing zygote thus carries fusion nucleus and female cytoplasm. The cytoplasmic organelles, chloroplast and mitochondria carries DNA which codes for genes involves in photosynthesis and transpiration. These vital process are directly linked with the yield and adaptation of a genotype to a particular environment. For this reason,  the cytoplasm of recurrent parent should be favored over donar in a cross. This is achieved by using the recurrent plant as the female, due to maternal  inheritance of cytoplasm.

Second reason is based on the fact that in any flowers, ovule is less abundant than pollens. A single flower will produce thousands of pollens but the ovules per flower is limited in number. The recombination process between homologous chromosomes of male and female parents in F1 gametic mother cells produces pollens and ovules with unique combination of male and female chromosomes. The pollens of F1 plants represents more variable combination of chromosomes than the ovules because of the abundancy of pollens. This will be explained by the following example of cotton

The cultivated cotton (Gossypium hirsutum L.) produces  an average of 30 seeds per flower. This indicates 30 ovules per flower. The same flower will produce 30,000 pollen grains.

Consider the reciprocal crosses between recurrent & donar by taking per single flower example

1. If F1 plant is used as the male and the inbred recurrent plant is used as the female

For the production of BC1F1 seeds : 30 uniform recurrent type ovules fuses with  30,000 segregating pollen grains = produce 30 BC1F1 seeds. Fertilization is a random process and all pollens has equal chance to unite with ovules. This increases the chance of desired recombinant pollen to get fertilized with the ovule. This chance events works equally in opposite directions as getting the undesired type combination is also same

2. If F1 plant is used as the female and the inbred recurrent plant is used as the male

For the production of BC1F1 seeds: 30 segregating ovules fuses 30,000 uniform pollen grains = produce 30 BC1F1 seeds. Here the recombination itself is limited to 30 ovules. The probability of obtaining desired recurrent plant type is very low.

In both crosses, the ovule number is the maximum limit of BC1F1 seeds. Getting desired type recombination in 30 pollens randomly selected out 30,000 pollens higher than getting desired type out of 30 ovules.

The summary is, 

1. The recurrent parent is used as the female due to the maternal inheritance of cytoplasm. This preserves the well adopted cytoplasm of female parent in subsequent crosses.

2. The segregating parent must always be used as male (Pollen source), which increase the probability of obtaining the desired recurrent plant types, to many folds

Note: Using of recurrent plant as female will  not be possible if back crossing is used for transferring Genetic male sterility genes (GMS). The donar is a male sterile plant. During F1 making, the recurrent parent will be used as male parent and the donar will used as female (sterile) parent.